BackgroundGlyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor.Methodology/Principal FindingsCultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1.Conclusions/SignificanceThe results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.
al. Development of a real-time reverse transcriptase PCR assay for type A infl uenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256-60. DOI: 10.1128/JCM.40.9.3256-3260.2002 Isolation of Brucella microti from SoilTo the Editor: Brucella microti is a recently described Brucella species (1) that was isolated in 2000 from systemically infected common voles (Microtus arvalis) in South Moravia, Czech Republic. The organism is characterized by rapid growth on standard media and high metabolic activity, which is atypical for Brucella (2). The biochemical profi le of B. microti is more similar to that of Ochrobactrum spp., of which most species are typical soil bacteria.On the basis of the close phylogenetic relationship of Brucella spp. and Ochrobactrum spp. and the high metabolic activity of B. microti, we hypothesized that this Brucella species might also have a reservoir in soil. To test this hypothesis, we investigated 15 soil samples collected on December 11, 2007, from sites in the area where B. microti was isolated from common voles in 2000 (2). Ten of the samples were collected from the surface and at a depth of up to 5 cm near different mouse burrows 5 m apart. The remaining 5 samples were collected from an unaffected area without clinical cases of vole infection. The pH of soil samples ranged from 5.9 to 6.3. No frosts were recorded before the time of collection.To specifi cally detect B. microti in soil samples, we have developed a PCR that targets a genomic island of 11 kb (H.C. Scholz et al., unpub. data) that is unique for B. microti. Briefl y, primers Bmispec_f (5′-AGATACTGGAACATAGCCCG-3′) and Bmispec_r (5′-ATACTCAGGC AGGATACCGC-3′) were used to amplify a 510-bp fragment of the genomic island. PCR conditions were denaturation at 94°C for 5 min, followed by 29 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Total DNA was prepared from 0.5 g of each soil sample by using the MO BIO Ultra Clean Soil DNA Kit (Dianova, Hamburg, Germany). DNA was eluted with 50 μL of double-deionized water of which 2 μL was used in PCRs. Template DNA of B. microti CCM 4915T was used as a positive control. Type strains of all recognized Brucella species, 1 strain of each biovar of all species, and type strains of 11 Ochrobactrum species were used as negative controls.In this PCR, 5 of 15 soil samples and the positive control were positive for the 510-bp fragment; other Brucella spp. and Ochrobactrum spp. were negative. Of the 5 positive samples, 3 were collected from surface soil collected near mouse burrows. However, the remaining 2 positive samples were collected from the unaffected and supposedly negative-control area.For direct cultivation of Brucella spp. from soil, 2 g each of 2 selected PCR-positive samples with the highest amplifi cation rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. Of a serial dilution in PBS (10 0 -10 -4 ), 100 μL was plated onto Brucella agar (Merck, Darmstadt, German...
From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.
Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.
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