ATP Bioluminescence Assay Research Articles

Abstract 964: Nicotinamide phosphoribosyltransferase (Nampt) regulates growth of non-small cell lung cancer cells with c-Met overexpression

Abstract Background: Non-small cell lung cancer (NSCLC) often has an oncogene addiction, including epidermal growth factor receptor (EGFR) signaling or hepatocyte growth factor (HGF)/c-Met signaling. NSCLC cells with these addictions require a large amount of intracellular ATP to activate their receptor tyrosine kinases (RTKs). Nicotinamide phosphoribosyltransferase (Nampt) is an enzyme to synthesize Nicotinamide adenine dinucleotide (NAD), which regulates cellular redox reactions as well as intracellular ATP biosynthesis. We have reported that the inhibition of intracellular Nampt function suppressed tumor growth via intracellular ATP reduction in EGFR-gene mutated NSCLC. We have developed a novel Nampt inhibitor, which is encapsulated within poly (lactic-co-glycolic acid) (PLGA) microspheres, to enhance the tumoricidal activity in vivo. We hypothesized that the Nampt inhibitor has a high anti-tumor activity in NSCLC with c-MET overexpression, and we evaluated the role of Nampt in growth of NSCLC with c-Met overexpression. Methods: In vitro study, expression of Nampt protein and mRNA in NSCLC cell lines (H1975, H1648, H820, H1993, from ATCC) were studied using western blotting and real-time RT-PCR (Lightcycler 2.0, Roche). NSCLC cells were treated with Nampt-siRNA (Invitrogen) or a Nampt inhibitor, FK866 (Axon medchem), and cell proliferation was examined using BrdU assay (Roche). Cell apoptosis in the NSCLC was detected by Flow cytometry (BD FACSAria II, BD Bioscience). Intracellular ATP levels in the treated NSCLC cells were also evaluated using bioluminescence assay (Roche). In western blotting, expression of c-MET signal proteins in the treated NSCLC was also studied. In vivo study, NSCLC cells were transplanted into 7-10 weeks-old female nude mice (CD-1Foxn1, Charles River), and the mice were treated with or without FK866. We used PLGA-FK866 in the tumor-bearing mice model. In immunohistochemistry, c-Met and MAPK signal proteins in xenograft tumors was evaluated using immunofluorescence microscopy (FV1000-D, OLYMPUS). Results: Nampt mRNA was overexpressed in all NSCLC cell lines. BrdU assay showed that treatment of Nampt-siRNA and FK866 suppressed cell proliferation and induced apoptosis in NSCLC cells. The expression of c-MET and MAPK signals were suppressed in FK866-treated NSCLC. ATP bioluminescence assay exhibited that ATP levels were decreased in FK866-treated NSCLC (p<0.05). In the tumor-bearing mice model, PLGA-FK866 suppressed growth of xenograft tumors (p<0.05), and achieved downregulation of MAPK signal proteins in xenografts. Conclusion: In the present study, we founded that the Nampt inhibitor suppressed NSCLC growth via suppression of intracellular ATP levels, and the expression of c-Met and MAPK signal proteins. These results suggest that Nampt has a potential to become a novel therapeutic target in NSCLC with c-MET overexpression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2011-964

Akt Requires Glucose Metabolism to Suppress Puma Expression and Prevent Apoptosis of Leukemic T Cells

The PI3K/Akt pathway is activated in stimulated cells and in many cancers to promote glucose metabolism and prevent cell death. Although inhibition of Akt-mediated cell survival may provide a means to eliminate cancer cells, this survival pathway remains incompletely understood. In particular, unlike anti-apoptotic Bcl-2 family proteins that prevent apoptosis independent of glucose, Akt requires glucose metabolism to inhibit cell death. This glucose dependence may occur in part through metabolic regulation of pro-apoptotic Bcl-2 family proteins. Here, we show that activated Akt relies on glycolysis to inhibit induction of Puma, which was uniquely sensitive to metabolic status among pro-apoptotic Bcl-2 family members and was rapidly up-regulated in glucose-deficient conditions. Importantly, preventing Puma expression was critical for Akt-mediated cell survival, as Puma deficiency protected cells from glucose deprivation and Akt could not readily block Puma-mediated apoptosis. In contrast, the pro-apoptotic Bcl-2 family protein Bim was induced normally even when constitutively active Akt was expressed, yet Akt could provide protection from Bim cytotoxicity. Up-regulation of Puma appeared mediated by decreased availability of mitochondrial metabolites rather than glycolysis itself, as alternative mitochondrial fuels could suppress Puma induction and apoptosis upon glucose deprivation. Metabolic regulation of Puma was mediated through combined p53-dependent transcriptional induction and control of Puma protein stability, with Puma degraded in nutrient-replete conditions and long lived in nutrient deficiency. Together, these data identify a key role for Bcl-2 family proteins in Akt-mediated cell survival that may be critical in normal immunity and in cancer through Akt-dependent stimulation of glycolysis to suppress Puma expression.

Resveratrol Potentiates Glucose-stimulated Insulin Secretion in INS-1E β-Cells and Human Islets through a SIRT1-dependent Mechanism

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.

EDTA chelation therapy in the treatment of toxic metals exposure

BACKGROUND: Metal induced toxicity with wide range of physiological, biochemical and behavioral dysfunctions was reported in many studies. The chelation has been used for treatment to toxic metals’ exposure for many years. In our current clinical study, we compared different chelation protocols and two forms of EDTA (sodium calcium edetate and sodium edetate) in treatments of toxic metal exposure. METHODS: A 24 h urine samples were collected from each subject before and after treatment by Ca-EDTA or Na-EDTA. The levels of toxic and essential metals were measured by Atomic Absorption Spectrometer (AAS) with graphite furnace and by Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES). The cellular levels of ATP were determined by ATP-bioluminescence assay. Mitochondrial potential was measured by fluorometer and flow-cytometer after cells’ staining by mitochondrial dye. RESULTS: Data from over 600 patients with a variety of complaints, but not acute toxic mineral exposure, given chelation therapy with sodium EDTA or calcium EDTA, were analyzed. Ca-EDTA and Na-EDTA at intravenous infusions of 3 g per treatment were equally effective in removing lead from the body, while Ca-EDTA was more effective in aluminum removal. The removal of lead was dose dependent but non-linear. Chelation by different doses of Na-EDTA (3 g and 1 g) resulted in mean difference in lead urine less than 50%. In addition, deficiencies in essential minerals correlated with greater pre-treatment lead and aluminum levels. The intrinsic toxicity of EDTA on cells was investigated. EDTA concentrations above 600 uM reduced cellular energy metabolism. CONCLUSION: Based on our data, we proposed that low dosages of chelating agents might be preferential for patients with no–occupational exposure, as the benefit of chelation is not linear with dosage, the risk of exposure to antidotes is increasing with increased dosages, and excretion of essential metals significantly increases with increased antidote dosage.

P11.19 The use of ATP bioluminescence assay as an educational tool to improve cleaning in ICU

P11.19 The use of ATP bioluminescence assay as an educational tool to improve cleaning in ICU

ATP-bioluminescence assay indicates the treatment of recurrent non-small cell lung cancer combined with malignant pleural effusion

This study was aimed to research the feasibility of ATP-bioluminescence assay (ATP-TCA) guiding the treatment on recurrent non-small cell lung cancer (NSCLC) combined with malignant pleural effusion. We collected 30 pleural fluid samples which were approved to be positive by cytology from recurrent NSCLC patients. These cells were cocultured with chemotherapy medicines, single agent or drugs combination. Five drug concentrations, two parallel holes were examined in vitro for 4 days, the results were measured by adding luciferase-fluorescein working system and luminescence analyzer. We applied chemotherapy medicines according to the results in vitro of ATP-TCA. There were differences among drug sensitivities of individuals. All the samples could be evaluated. Effective single drugs included cisplatinum, mitomycin C, doxorubicin, and pemetrexed disodium; sensitive drugs in the combination therapy were gemcitabine plus cisplatin, vinorelbine plus cisplatin, paclitaxel plus cisplatin, docetaxel plus cisplatin, and mitomycin C, vindesine plus cisplatin, in which gemcitabine + cisplatin (GEM + DDP) in vitro was the most efficient program. ATP-TCA in vitro sensitivity assay is rapid, reliable, and simple to guide the treatment of recurrent NSCLC with malignant pleural effusion, and can help clinicians to make the individual chemotherapy program.

Comparison of potency assays using different read-out systems and their suitability for quality control

In this study, three of the most commonly used non-radioactive read-out systems for bioassays, the tetrazolium salt MTS/PMS, the fluorescent dye Alamar Blue and the ATP bioluminescence assay were compared regarding their suitability for quality control purposes. In this regard, three different potency assays using murine CTLL-2, as well as human DiFi and Kit 225 cells were performed. No major differences regarding accuracy and precision were detected between the different read-out systems. Both workload and hands-on time were similar for all three assays used. All read-out systems were suitable for use in quality control. However, luminescence and fluorescence techniques were much more sensitive than the colorimetric system. The first two could determine approximately ten times lower drug concentrations, and the assay could be performed by using considerably lower cell numbers. Moreover, in two of the three potency assays, the luminescence and fluorescence read-out systems provided higher signal to noise ratios leading to a higher precision of the assays. Regarding the comparison of the luminescence and fluorescence system, the ATP assay has the advantage to be much faster than the Alamar Blue assay. Consequently, in this study, the luminescence technique turned out to be the most advantageous of the three read-out systems.

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10 3 cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1–5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Basic peptide protamine exerts antimicrobial activity against periodontopathic bacteria——Growth inhibition of periodontopathic bacteria by protamine

Protamine was investigated for its antibacterial activity against the periodontal pathogens, Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. We determined the minimum inhibitory concentrations of protamine and its hydrolysate and their bactericidal activity. Protamine inhibited the growth of all periodontopathic bacteria tested on agar plates. Protamine, which MIC was 6.3 × 10-7 g L-1, was most effective against P. gingivalis. The antibacterial effect of native protamine was higher than that of its hydrolysate. An ATP bioluminescence assay revealed that protamine showed bactericidal activity against P. gingivalis in a time-dependent manner. These results indicate that protamine could be candidate peptide for prevention of P. gingivalis infection.

O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

Implications On Drug Resistance and Survival of ABCB1 Single Nucleotide Polymorphisms in Normal Karyotype De Novo AML.

Abstract 2648Poster Board II-624 Background:Multidrug resistance and expression of the ATP-dependent drug transporting protein ABCB1 is a clinically relevant problem in the treatment of acute myeloid leukaemia. Several single nucleotide polymorphisms (SNPs) in the ABCB1 have been associated with altered P-glycoprotein expression and phenotype. These SNPs might influence the clinical outcome in AML and predict individual differences in response to therapy with ABCB1 substrates. Aims:To investigate the impact of the ABCB1 SNPs in exon 11, 12, 21 and 26 on treatment response, survival and in vitro drug sensitivity in AML patients. Methods:PCR and Pyrosequencing were used to determine the genotype of the SNPs G1199T/A, C1236T, A1308G, G2677T/A and C3435T in 100 de novo AML patients with normal karyotype treated at Linköping University Hospital or Karolinska University Hospital. Almost all patients were treated with one anthracycline and Ara-C during the induction regime. The affect of the genetic variants in ABCB1 on survival were analysed by Kaplan-Meier Log-rank tests and multivariant analysis by Cox regression. Patients receiving transplantation were censored at that point in the analysis. A Nordic reference material of 400 healthy volunteers of equal age and sex distribution was also included. NPM1 and FLT3-ITD mutations were determined by PCR. Leukemic cells were isolated and drug sensitivity was measured after 4 days culturing by a bioluminescence ATP-assay. Results:The survival of the AML patients was significantly correlated to the ABCB1 genotypes C1236T (P=0.02) and G2677T (P=0.02), with a borderline significance for G1199A (p=0.06). For the C1236T SNP the mean survival was 0.7, 1.3 and 1.8 years for the wild type, heterozygous and homozygous variants, respectively. The mean survival for patients with G/G, G/T and T/T genotype of SNP G2677T/A was 0.7, 1.2 and 1.7 years, respectively. Only the wild type of A1308T was found in the material and C3435T did not correlate to survival. Multivariate analysis showed that 1236T/T and 2677T/T were independent factors for survival (hazard ratio 0.24 and 0.22). Comparison of allele frequencies between AML patients and healthy volunteers showed no significant difference. In vitro testing showed that leukemic cells from patients carrying 1236T/T or 2677T/T were significantly more susceptible for mitoxantrone and borderline susceptible to daunorubicine and etoposide, substrates for Pgp but not to Ara-C. Conclusions:Our findings suggest that ABCB1 SNPs do not affect the development of the disease but the survival after chemotherapy possibly by impact on drug sensitivity. The correlation between ABCB1 genotype and the overall survival of AML patients might provide useful information for treatment strategies and individualized chemotherapy. Disclosures:Paul:Aprea AB: Consultancy, Research Funding.

Effect of eel galectin AJL-1 on periodontopathic bacterial biofilm formation and their lipopolysaccharide-mediated inflammatory cytokine induction

Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, infectious pathogenic bacteria found in oral biofilm, cause periodontal disease. The inhibitory effect of AJL-1, a galectin present in the skin mucus of the Japanese eel Anguilla japonica, on biofilm formation by each of these strains was investigated by staining adherent bacteria on culture plates with crystal violet. An ATP bioluminescence assay was used to determine whether inhibition of biofilm formation was due to the bactericidal activity of AJL-1. The effect of AJL-1 on cytokine induction in human umbilical vascular endothelial cells (HUVECs) by lipopolysaccharide (LPS) isolated from A. actinomycetemcomitans was also investigated by enzyme-linked immunosorbent assay (ELISA). AJL-1 significantly inhibited biofilm formation by A. actinomycetemcomitans strains Y4, ATCC 29523 and ATCC 29524 but not by any strain of P. gingivalis or P. intermedia, and showed no bactericidal activity against A. actinomycetemcomitans strains. AJL-1 markedly suppressed interleukin (IL)-6 and IL-8 induction in HUVECs by LPS from A. actinomycetemcomitans strains Y4 and ATCC 29523. These observations indicate that AJL-1 is an effective inhibitor of biofilm formation by A. actinomycetemcomitans as well as of inflammatory cytokine induction in HUVECs by LPS. These finding indicate that AJL-1 may be of therapeutic value in A. actinomycetemcomitans-associated periodontal diseases.

Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coli FoF1-ATP Synthase

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

A non-toxic herbal remedy which enhance lymphocyte activity and cytokine secretion: Ganoderma lucidum

Ganoderma lucidum is grown in the deep foliage of South-western China, where it is free from heavy metals and pollutions from populated cities. G. lucidum contains biologically active components, polysaccharides, which have been shown to enhance the immune system. In China, G. lucidum is commonly used to treat cancer, allergies, inflammation, hypertension and platelet-aggregation. This study investigates the proliferation, metabolism and cytokine secretion of lymphocytes in vitro upon G. lucidum stimulation. The powder of G. lucidum was provided by Mei Shan Tang Limited (Hong Kong) and tested to be free of heavy metals and microorganisms. Lymphocytes were extracted from 66 healthy human subjects and cultured with a pre-determined optimal concentration of G. lucidum (50-100 ug/mL) or a control medium for 72 h. Proliferation was measured with cell proliferation reagent, and metabolic rate was measured with ATP bioluminescence assay. Levels of IL-2, IL-4, IL-8, IL-10, GM-CSF and TNF-
were measured with ELISA. Meanwhile, toxicity was tested by feeding 70 rats with G. lucidum. No significant toxicity was detected in the rats. In human lymphocytes, G. lucidum was found to significantly increase cell proliferation and metabolism (p cellular immunity. G. lucidum is therefore potential candidate as a safe immunostimulant for patients on antin-eoplastic therapy.

Perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorização das condições de higiene em uma linha de produção de queijo de coalho

Avaliou-se o perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorou-se as condições de higiene em uma linha de produção de queijo de coalho por meio da técnica de bioluminescência (ATP). A população de Staphylococcus sp. variou de <1UFC mL-1, no leite pasteurizado a 1,5 x 10(7)UFC mL-1 no leite cru, enquanto que a de Staphylococcus coagulase-positiva oscilou entre <1UFC mL-1 no leite pasteurizado e 5,0 x 10(6)UFC mL-1 no leite cru. Staphylococcus coagulase-positiva foi detectada em 100% (25/25) das amostras de leite cru e em 8% (2/25) das amostras de queijos. Foram identificadas 12 espécies de Staphylococcus entre os 68 isolados selecionados, sendo nove coagulase negativa e três coagulase-positiva. No leite cru observou-se alta freqüência de Staphylococcus coagulase positiva, principalmente S. aureus, enquanto que em leite pasteurizado, coalhada e queijo verificou-se alta freqüência de Staphylococcus coagulase negativa e baixa freqüência de Staphylococcus coagulase-positiva. A presença de enterotoxina estafilocócica foi constatada em 20% das amostras de leite cru e, conseqüentemente, também foi detectada no leite pasteurizado, na coalhada e no queijo produzido. A dosagem de ATP permitiu avaliar o estado de higienização das superfícies, o qual foi considerado: adequado em 62,1% (36/95), "estado de alerta" em 23,2% (22/95) e inadequado em 14,7% (14/95) das superfícies. A detecção de espécies de Staphylococcus com potencial enterotoxigênico e a presença de enterotoxinas revelam a disseminação da contaminação na linha de produção do queijo de coalho provavelmente devido a falhas na aplicação das Boas Práticas de Fabricação desde a obtenção da matéria-prima até a produção final do queijo.

Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents

Fungal contamination is a major cause of deterioration in libraries and archives. Curators and conservators increasingly need rapid microbiological analyses. This paper presents a rapid detection method for the fungal contaminants on documents. A previous study showed that the calculation of energy charge, using bioluminescence ATP assays, provides a useful indicator to determinate the viability of fungal strains. We argue that this sensitive and time-saving method is better than traditional culture techniques. However, the procedure needs to be modified to make it usable for lay persons. An improved and simplified protocol is proposed here for the extraction of adenylate nucleotides (AN) from fungal spores and for their measurements. Our new procedure can detect the existence of viable fungal strains on documents, presenting suspect spots within minutes. The extraction is performed by filtration with DMSO-TE solution as extractant. The different step of the measurement of AN content is carried out successively in a single test tube instead of the three tubes necessary in the initial method. The new procedure was tested on 12 strains among those most frequently found in archives and libraries and validated on swab samples from real documents.

Rapid Antimicrobial Susceptibility Determination of Uropathogens in Clinical Urine Specimens by Use of ATP Bioluminescence

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

Mitochondrial ND5 Gene Variation Associated with Encephalomyopathy and Mitochondrial ATP Consumption

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.