A novel and efficient assay for identification and quantification of Acidithiobacillus ferrooxidans in bioleaching samples

Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic, and chemolithotrophic bacterium that is active in bioleaching. The leaching efficacy is directly influenced by the biomass changes of this specie in bioleaching microbial community. In order to perform a simple and sensitive assay on A. ferrooxidans from mixed strains in this process, a novel assay was developed based on sandwich hybridization assay with the aid of S1 nuclease treatment and fluorescent labeling. In the work, a designed DNA probe complementary to the conservative region of its 16S rRNA was synthesized, which showed high accuracy for distinguishing homologous species with the exclusion of even-only two base pairs difference. The specificity of this assay was verified in different systems with mixed strains, and the quantitative result was proved by comparison of microscopic cell counting. The detection sensitivity was about 8 × 10(2) cells/ml and the inter-assay coefficient of variation of three independent assays was from 3.8 to 7.7 %, respectively. In addition, the cycle of assay was about 3-4 h when the cost estimated was less than $0.5 per sample. This assay method might be applied for identifying and monitoring any kind of bacterial strain from a mixed microbial flora in bioleaching or other areas.     

MutS-mediated enrichment of mutated DNA produced by directed evolution in vitro

Directed evolution in vitro is a powerful tool in the study and design of protein function. However, screening the desired mutants is a difficult task. To facilitate the screening, a method is proposed to eliminate wild type sequences and increase mutated DNA sequences, which is based on the preferential binding of MutS protein to heteroduplex DNA. Following error-prone PCR, amplified products are denatured and re-annealed to form heteroduplex and homoduplex DNA. Heteroduplexes are selectively bound to an engineered MutS protein and immobilized on a Strep-Tactin column. Homoduplexes are effectively removed by washing, and the final elution is enriched in mutated DNA sequences. One round of mutated DNA enrichment resulted in an about 2.3-fold of increase in mutation frequency compared to the control. The percentage of mutants rose from 44% in the control sample to 72% in the enrichment sample. Fluorescent assay by flow cytometry showed that the enrichment method increased the mutants with changed fluorescent activity by about 2.2-fold, which strongly justified the efficiency of enrichment in increasing mutants with functional changes. With reduced workload of screening and increased possibility of obtaining mutants with functional changes, the overall efficiency was improved by MutS-mediated enrichment of mutated DNA.     

PCR法在小鼠早期念珠菌病诊断中的应用研究

目的探讨PCR法对早期念珠菌病的诊断价值。方法采用Biospin真菌基因组DNA提取试剂盒提取感染小鼠全血白色念珠菌DNA,并与血培养和脾脏、肾脏组织病理检查结果比较。结果白色念珠菌标准菌株和两株临床分离菌株经PCR测定后,均可扩增到分子量大小约为500bp的特异性条带。在小鼠早期念珠菌病的感染中,运用PCR法较血培养和组织病理检查敏感性高。结论PCR法是一种快速、灵敏且特异性高的检查手段,为临床早期检测真菌病提供实验依据。     

Effects of Drugs Interfering with Dopamine and Noradrenaline Biosynthesis on the Endogenous 3,4- Dihydroxyphenylalanine Levels in Rat Brain

A chemical assay of 3,4-dihydroxyphenylalanine (DOPA) in nervous tissue is described. The method is based on a rapidly performed isolation of DOPA on small Sephadex G-10 columns, followed by reverse-phase HPLC with a trichloroacetic acid-containing eluent, in conjunction with a rotating disk electrochemical detector. The detection limit of the assay (about 100 pg/tissue sample) permits a detailed investigation of the regional distribution of endogenous DOPA levels in the rat brain. DOPA as well as dopamine (DA) could be quantified in the same chromatographic run. The assay was applied to a study of the effects of alpha-methyl-p-tyrosine, apomorphine, chlorpromazine, clonidine, gamma-butyrolactone, haloperidol, morphine, oxotremorine, pargyline, reserpine, and tyrosine methylester on the concentration of DOPA in the striatum, hypothalamus, frontal cortex, and cerebellum of the rat brain. Drugs known to interact with DA biosynthesis all caused characteristic changes of the DOPA content in the striatum and not in nondopaminergic brain areas. A close correlation existed between drug-induced changes in tyrosine hydroxylase activity and changes in the DOPA content in the striatum. Tyrosine methylester increased DOPA concentrations in all brain areas studied.     

Structural control of endoplasmic reticulum-associated degradation: effect of chemical chaperones on 3-hydroxy-3-methylglutaryl-CoA reductase

The endoplasmic reticulum (ER) quality control pathway destroys misfolded and unassembled proteins in the ER. Most substrates of this ER-associated degradation (ERAD) pathway are constitutively targeted for destruction through recognition of poorly understood structural hallmarks of misfolding. However, the normal yeast ER membrane protein 3-hydroxy-3-methylglutaryl-CoA reductase (Hmg2p) undergoes ERAD that is physiologically regulated by sterol pathway signals. We have proposed that Hmg2p ERAD occurs by a regulated transition to an ERAD quality control substrate. Consistent with this, we had previously shown that Hmg2p is strongly stabilized by chemical chaperones such as glycerol, which stabilize misfolded proteins. To understand the features of Hmg2p that permit regulated ERAD, we have thoroughly characterized the effects of chemical chaperones on Hmg2p. These agents caused a reversible, immediate, direct change in Hmg2p degradation consistent with an effect on Hmg2p structure. We devised an in vitro limited proteolysis assay of Hmg2p in its native membranes. In vitro, chemical chaperones caused a dramatic, rapid change in Hmg2p structure to a less accessible form. As in the living cell, the in vitro action of chemical chaperones was highly specific for Hmg2p and completely reversible. To evaluate the physiological relevance of this model behavior, we used the limited proteolysis assay to examine the effects of changing in vivo degradation signals on Hmg2p structure. We found that changes similar to those observed with chemical chaperones were brought about by alteration of natural degradation signal. Thus, Hmg2p can undergo significant, reversible structural changes that are relevant to the physiological control of Hmg2p ERAD. These findings support the idea that Hmg2p regulation is brought about by regulated alteration of folding state. Considering the ubiquitous nature of quality control pathways in biology, it may be that this strategy of regulation is widespread.     

A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.     

Fluorometric measurement of nitrite/nitrate by 2,3-diaminonaphthalene

We describe a step-by-step protocol for measuring the stable products of the nitric oxide (NO) pathway: nitrite, nitrite plus nitrate and nitrate. This described protocol is easy to apply and is about 50 times more sensitive than the commonly used Griess reaction or commercially available assay kits based on the Griess reaction. It also allows the study of minimal changes in the NO pathway. With this method, it takes about 3 h to analyze the above-mentioned stable products in culture supernatants or in various body fluids, and the method has a sensitive linear range of 0.02-10.0 microM. This restricted linear range suggests that the technique is useful for studying small changes of nitrite and nitrate, rather than for routine diagnostic measurements.     

Epidemiologic evidence for a role of telomere dysfunction in cancer etiology

Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10μM benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10μM) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1μM and 10μM BaP-treated groups, 2 in the 0.1μM and 10μM BaP-treated groups, 4 in the 0.1μM and 1μM BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical-host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.     

软骨多糖诱导MCF-7乳腺癌细胞凋亡的实验研究

研究软骨多糖诱导MCF-7乳腺癌细胞凋亡及其作用机理。方法:选用MCF-7人类乳腺癌细胞系体外培养,应用MTT法检测细胞生长抑制率,TUNEL法检测细胞凋亡率,HE染色法观察细胞形态学改变,流式细胞仪检测细胞周期的变化,免疫荧光方法检测BCL-2BAD及波形蛋白Vimentin的表达率。结果:软骨多糖对MCF-7细胞体外生长具有明显的抑制作用,且呈时间和浓度依赖性;软骨多糖可诱导MCF-7细胞发生凋亡并伴随有凋亡小体出现等形态学变化;软骨多糖促进BCL-2蛋白的表达水平下降,BAD表达水平上升,及Vimentin的降解。结论:软骨多糖能够在体外诱导MCF-7细胞凋亡,是一种新型的抗乳腺癌活性物质。     

Hypertonic saline enhances expression of phosphorylated histone H2AX after irradiation

Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called "foci" that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na(+) increased gammaH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of gammaH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced gammaH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that gammaH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.     

小鼠骨髓瘤中肿瘤干细胞的初步分析

探讨小鼠骨髓瘤(SP2/0细胞)中肿瘤干细胞存在与否。以克隆形成试验检测SP2/0细胞中具有形成克隆能力细胞的大体比例;采用BrdU标记滞留试验检测SP2/0细胞中含有DNA永生化链的细胞,即具有干细胞特性的细胞;检测SP2/0细胞中具有干细胞特性的SP细胞存在情况及其比例。结果显示,SP2/0细胞中有一部分细胞具有形成克隆的能力;SP2/0细胞中含有DNA永生化链的细胞;SP2/0细胞中存在SP细胞,其比例约为0.7%。而且SP2/0细胞中存在肿瘤干细胞。     

Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells

The present study was undertaken to standardize a dichlorofluorescein (DCF) assay for measurement of radiation-induced oxidation of dichlorofluorescin (DCFH) substrate in MCF-10 cells. This assay was highly sensitive and capable of detecting increased DCFH oxidation in the cells exposed to gamma radiation at doses as low as 1.5 cGy with linear dose-response curves. However, the slope of the dose-response curves varied considerably from one experiment to another and was influenced by the fluorescent substrate concentration and cell density. To make the assay reproducible so that results obtained from different experiments could be compared, a series of conversion factors and equations have been established to normalize the data for these variables. The results demonstrate that the DCF assay, as standardized in the present study, is highly reproducible with acceptable assay precision. The normalized results can be compared from one experiment to another even when the experiments were performed using different fluorescent substrate concentrations and/or cell densities. Since changes in DCFH oxidation may be related to changes that are indicative of oxidative stress in cells, this assay can be useful to quantify radiation-induced oxidative stress and evaluate the efficacy of antioxidant agents in protection against radiation-induced oxidative stress.     

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.     

The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize (Zea mays).

Prolamellar bodies (PLB) contain two photochemically active forms of the enzyme protochlorophyllide oxidoreductase POR-PChlide640 and POR-PChlide650 (the spectral forms of POR-Chlide complexes with absorption maxima at the indicated wavelengths). Resuspension of maize PLB in media with a pH below 6.8 leads to a rapid conversion of POR-PChlide650 to POR-PChlide640 and a dramatic re-organization of the PLB membrane system. In the absence of excess NADPH, the absorption maximum of the POR complex undergoes a further shift to about 635 nm. This latter shift is reversible on the re-addition of NADPH with a half-saturation value of about 0.25 mm NADPH for POR-PChlide640 reformation. The disappearance of POR-PChlide650 and the reorganization of the PLB, however, are irreversible. Restoration of low-pH treated PLB to pH 7.5 leads to a further breakdown down of the PLB membrane and no reformation of POR-PChlide650. Related spectral changes are seen in PLB aged at room temperature at pH 7.5 in NADPH-free assay medium. The reformation of POR-PChlide650 in this system is readily reversible on re-addition of NADPH with a half-saturation value about 1.0 microm. Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide650, membrane organization and NADPH binding. The low-pH driven spectral changes seen in maize PLB are shown to be accelerated by adenosine AMP, ADP and ATP. The significance of this is discussed in terms of current suggestions of the possible involvement of phosphorylation (or adenylation) in changes in the aggregational state of the POR complex.     

Kinetics of ATP/ADP binding to the gp16 ATPase

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 μM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.     

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.     

High-throughput assay for small molecules that modulate zebrafish embryonic heart rate

To increase the facility and throughput of scoring phenotypic traits in embryonic zebrafish, we developed an automated micro-well assay for heart rate using automated fluorescence microscopy of transgenic embryos expressing green fluorescent protein in myocardium. The assay measures heart rates efficiently and accurately over a large linear dynamic range, and it rapidly characterizes dose dependence and kinetics of small molecule-induced changes in heart rate. This is the first high-throughput micro-well assay for organ function in an intact vertebrate.     

MTT法用于药物对L2细胞毒性的实验研究

目的：评价噻唑蓝（MTT）法检测药物对细胞的毒性作用的可靠性。方法：大鼠肺泡上皮L2细胞以叔丁基对苯二酚（TBHQ）10．100μM,BsO以1-10mM分别处理,用MTT法检测细胞活性、JC-1（5,5’,6,6’-四氯．1,1’,3,3’-四乙基苯并咪唑羰花青碘化物）荧光染料法检测细胞线粒体电位改变、台盼蓝排斥实验检测细胞死亡率,分析各指标的情况。结果：在处理剂量范围,MTT法检测到的光密度（OD）值未能达到一般判断的半数抑制浓度（ic50）水平,最高抑制率仅达到30％左右;台盼蓝排斥试验检测数据表明TBHQ的LC50值为50μM,丁硫氨酸亚砜胺（BSO）为5mM;利用JC-1荧光染料判断的半数凋亡剂量分别为50μM和7mM。结论：MTT法作为最常采用的细胞生长抑制检测手段,但在某些特定实验中可能不能客观地反映细胞的活性,建议多种方法结合进行评价。     

Influence of chronic inflammation on the malignant phenotypes and the plasticity of colorectal cancer cells

Sporadic adenoma or adenocarcinoma is often detected during endoscopic surveillance of patients with ulcerative colitis (UC). However, it is occasionally difficult to distinguish these neoplasms from dysplasia or colitis-associated cancers because of the influence of inflammation. However, the influence of inflammation on sporadic neoplasms is not well characterised. To assess this influence, we established a long-term inflammation model of colon cancer cells by inflammatory stimulation with tumour necrosis factor-α, flagellin and interleukin-1β for 60 weeks. Then, the malignant phenotypes were evaluated using the MTS assay, Annexin V fluorescence assay, cell migration assay and sphere formation assay. The influence of P53 function on these phenotypes was assessed with a TP53 mutation model using the CRISPR/Cas9 system. A long-term inflammation model of LS174T cells was established for the first time with continuous inflammatory signalling. Chronic inflammation induced apoptosis and suppressed the proliferation and stemness of these cancer cells via the action of P53. It also enhanced the invasiveness of LS174T cells. Moreover, these phenotypic changes and changes in inflammatory signalling were recoverable after the removal of inflammatory stimuli, suggesting that colon cancer cells have higher plasticity than normal intestinal epithelial cells. In conclusion, our results suggest that sporadic neoplasms in patients with UC are affected by chronic inflammation but are not essentially altered.     

A new reporter gene system suited for cell-free protein synthesis and high-throughput screening in small reaction volumes.

The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K+ (and Rb+). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively.