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1.
ATP methodology needs to be further standardized and improved in order to avoid the pitfalls that have sometimes hampered its application to biomass assays. The following steps have been reconsidered as far as the bacteriological applications is concerned:
- destruction of free and somatic ATP: replacement of apyrase by mammalian ATPase, more readily accessible to specific inhibition;
- extraction of bacterial ATP: protection of luciferase by lipids against inhibitory effect of cationic detergents with production of a constant light response.
2.
The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin. 相似文献
3.
Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity 总被引:1,自引:0,他引:1
Khan HA 《Journal of biosciences》2003,28(4):379-382
Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate
(ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based onin vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results
showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid
extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction. 相似文献
4.
An adenosine triphosphate (ATP) bioluminescence‐based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish‐red stains, damaging the aesthetics of documents and their long‐term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold‐like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm2, suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm2. ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
5.
Tetsuya Satoh Kosuke Tsuruta Yasuharu Shinoda Ryuichi Hirota Kenichi Noda Akio Kuroda Yuji Murakami 《Analytical biochemistry》2009,395(2):161-165
We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed. 相似文献
6.
The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F1 portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pHi (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pHi of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pHi homoeostasis. 相似文献
7.
A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture. 相似文献
8.
We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay. 相似文献
9.
What determines the intracellular ATP concentration 总被引:1,自引:0,他引:1
Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfilling useful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools. 相似文献
10.
V. V. Borisova I. A. Pyshnaya D. V. Pyshnyi L. A. Frank 《Russian Journal of Bioorganic Chemistry》2008,34(6):709-715
The recombinant Ca2+-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample. 相似文献
11.
A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively. 相似文献
12.
The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined. 相似文献
13.
A sensitive method for the analysis of ATP and phosphocreatine (PCr) in single human skeletal muscle fibres is described. Muscle tissue was freeze-dried and single fibres were dissected free with the aid of low-power microscopy. The fibres were then extracted in trichloroacetic acid and neutralized with KHCO3. The assay is based on the continuous monitoring of light produced as a result of ATP degradation in the firefly luciferase reaction. PCr is measured as the amount of ATP formed in the creatine kinase reaction. The coefficient of variation was less than 4% for both ATP and PCr determination. The amount of tissue required for the assay is approximately 0.5 μg (dry weight). The assay showed good agreement with spectrophotometric and high-performance liquid chromatographic (HPLC) measurements made upon extracts of whole muscle tissue. 相似文献
14.
Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes 总被引:4,自引:0,他引:4
Cheng G Polito CC Haines JK Shafizadeh SF Fiorini RN Zhou X Schmidt MG Chavin KD 《Biochemical and biophysical research communications》2003,308(3):573-580
Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury. 相似文献
15.
16.
A novel technique has been developed for semiquantitative detection of glutathione (GSH) in small volumes of liquid samples. GSH is detected via enzymatic linkage to the NADP/NADPH + H+ redox system through glutathione reductase. Accumulated NADPH is measured via the bioluminescent FMN oxidoreductase bacterial luciferase reaction. A linear correlation is obtained between bioluminescence intensity of the luciferase reaction and the GSH content of the liquid sample. Possible applications of this procedure are discussed. © 1998 John Wiley & Sons, Ltd. 相似文献
17.
本文从快速检测的必要性出发,介绍了ATP生物荧光法及其原理,并针对某款化妆品通过一系列实验建立了一种快速检测菌落总数的方法。将该方法用于CTFA(美国化妆品香料香精协会,Cosmetic Toiletry and Fragrance Association)实验中,并通过与平板计数法结果进行比对,发现该方法可以有效的运用于样品细菌总数的快速检测。 相似文献
18.
Michael Dattilo Nicholas J. Penington Keith Williams 《Journal of cellular physiology》2020,235(10):7056-7066
TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel level using patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels. 相似文献
19.
20.
Jidong Yan Juan Tian Yuewen Zheng Yan Han Shemin Lu 《Cell biochemistry and function》2012,30(8):657-663
Selenium (Se) is an essential micronutrient, and low Se intake in Se‐deficient areas plays roles in an endemic osteochondropathy characterized by chondronecrosis in growth plate and articular cartilage. However, the biological activities of Se on cartilage are largely unknown. In this study, we examined the effects of Se on chondrogenic cell ATDC5 and the possible mechanisms involved. We demonstrated that Se stimulated ATDC5 cell proliferation under serum deprivation but not routine culture. Furthermore, Se promoted G1‐phase cell cycle progression along with induction of cyclin D1 expression at the mRNA and protein level. Moreover, Se increased intracellular ATP content and decreased intracellular superoxide anion concentration without affecting intracellular redox status as estimated by ratio of the reduced and oxidized glutathione. In addition, suppression of intracellular ATP synthesis by glycolysis inhibitor or mitochondrial uncoupler both abrogated Se‐mediated cyclin D1 induction. These findings suggest Se stimulates proliferation of chondrogenic cell ATDC5 through acceleration of cell cycle progression accompanied with cyclin D1 induction by enhancement of intracellular ATP content. This novel finding provides evidence for a role of Se in cartilage formation and degenerative processes and further supports the relationship between Se status and cartilage function that may lead to better utilization of Se for cartilage homeostasis. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献