Abstract: We developed a novel filtration-based method that can eliminate dead or severely damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p<0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R(2)>0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.
Abstract: The toxicities of three plant volatiles, (2E)-hexenal, (2E, 6Z)-nonadienal and (2E)-nonenal, intermediate products of the oxylipin biosynthesis pathway, were tested on three mites of importance for medical purposes and as pests. The aldehydes were diluted in hexane separately and incorporated into diets in ranges of 4-143 mg g(-1). The final density of mites in control and aldehyde-enriched diets was compared after 21 days. The aldehydes were toxic to the mites, whose final density showed an inverse correlation with aldehyde concentration. In addition to the effects of aldehyde concentration, the final density of mites was also influenced by the different aldehydes tested and the interaction among aldehyde concentration and chemical structure. In a functional combination of aldehydes and species, the doses calculated for growth inhibition and eradication of mites ranged from 4 to 35 mg g(-1) and from 36 to 314 mg g(-1), respectively. Due to the protective role displayed by natural six-carbon and nine-carbon aldehydes, these compounds are potential candidates for controlling stored-product mites in stored food and feed products.
Abstract: BACKGROUND: In these years a huge number of human transcripts has been found that do not code for proteins, named non-protein coding RNAs. In most cases, small (miRNAs, snoRNAs) and long RNAs (antisense RNA, dsRNA, and long RNA species) have many roles, functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, and controlling protein ubiquitination and degradation. Various species of npcRNAs have been found differentially expressed in different types of cancer. This review discusses the published data and new results on the expression of a subset of npcRNAs. CONCLUSION: These results underscore the complexity of the RNA world and provide further evidence on the involvement of functional RNAs in cancer cell growth control.
Abstract: Dot blot and deoxyribonucleic acid (DNA) array hybridization assays for the traceability of Lactobacillus species in food have been developed to monitor and validate typical food products. A primer set was designed to amplify the 540-bp region located at +157 of the tuf (Elongation factor Tu) gene of the Lactobacillus genus. An oligonucleotide array, containing 73 Lactobacillus species-specific tuf sequences representing 21 species, was developed and tested for identifying L. paracasei, L. rhamnosus, L. plantarum, and L. buchneri. We also tested a rapid screening method for monitoring the species present in airy samples. Dot blot hybridization identified polymerase chain reaction amplicons immobilized on nylon membranes, using six tuf-based cyanine-3-labeled 18-mer oligonucleotides, specific for L. paracasei, L. zeae, L. fermentum, L. plantarum, L. rhamnosus, and L. buchneri. This method discriminates between multiple species of Lactobacilli isolated directly from cheese samples, simultaneously. The tuf gene sequences, verified here with the DNA array method and used in dot blot hybridization, were shown to be a reliable tool for the simultaneous detection and differentiation of four Lactobacillus species. The hybridization techniques developed in this study may be useful in food processing and the analysis of food origin traceability.
Abstract: In this work, purification of lactoferrin from whey was performed with high recovery rate. Lactoferrin was then exploited in the preparation of food emulsions. Two tertiary emulsions, formed by olive oil, lecithin, chitosan, and lactoferrin, were compared: both the emulsions showed similar turbidity and stability. In the secondary emulsion formed by oil/lecithin/chitosan, the pH was increased to 9 before addition of lactoferrin. Then, lactoferrin was added, and the pH was stabilized above pH 9. Lactoferrin was found in amounts of 1 to 2.5 mg/ml in the multiple experiments. A fraction of the added lactoferrin was also present in a milky layer above the emulsion layer. This was, to our knowledge, the first study of emulsions made exploiting the interactions between lactoferrin and chitosan. It was noted that chitosan droplets remained soluble, although the hydrocolloid solubility occurs at pH lower than 5.9. These results showed the feasibility of manufacturing lactoferrin-based emulsions as functional foods.
Abstract: In this study label-free a SPR immunosensor and a protein array based methods were used for their application in bacterial detection. L. monocytogenes, one of the most difficult to treat bacteria, was used as model pathogen. While the use of DNA arrays for bacteria detection and identification is largely documented, no studies are available on protein array (PA) based bacteria detection. In protein array approach an affinity-purified monoclonal antibody was used as capture antibody. Protein array detection limit was of 102-103CFU/mL. SPR immunoassays were prepared by chemically binding L. monocytogenes cells on a proper gold substrate. After immobilization of antigen on gold substrate, a further incubation with anti-L. monocytogenes antibodies was performed. The technique was revealed as powerful and fast method for the monitoring of binding between the investigated antigen and antibodies. On the other side, array based methods, although more expensive due to the labelling step, can be used with a set of species-specific antibodies allowing for simultaneous detection of multiple pathogens in a single hybridization step in no more then 2.5 h.
Abstract: Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.
Abstract: New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.
Abstract: Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.
Abstract: In recent years, public pressure to reduce the use of synthetic fungicides in agriculture has increased. Concerns have been raised about both the environmental impact and the potential health risk related to the use of these compounds. Therefore, considerable efforts have been made towards the development of alternative crop protectants. The European Commission has been actively encouraging the development and commercial implementation of new compounds known as 'green chemicals'. In this context, an increase in the knowledge of plant defence responses to toxigenic fungi, which is covered in this review, will help to discover new plant products with antifungal activity and to design new strategies to improve plant resistance to these pathogens.
Abstract: BACKGROUND: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. METHODS: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. RESULTS: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. CONCLUSIONS: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.
Abstract: Twenty-nine strains of Lactic Acid Bacteria isolated from the typical Pecorino cheese of the Salento area of Italy, were identified and grouped according to their genetic similarity. A preliminary characterisation of the strains was conducted by means of morphological and biochemical analysis, but molecular approaches were necessary for the clear identification of the species. For the species detection, the amplification and sequencing of the 16S rDNA gene was employed In addition, restriction analysis of amplified rDNA (ARDRA) and PCR and AFLP fingerprinting enabled inter- and intra-specific variation to be estimated UPGMA cluster analysis was used to divide the strains into distinct clusters which corresponded with the species delineation obtained by molecular identification. The data obtained show that the community of lactobacilli responsible for the fermentation and aging of Pecorino cheese is composed of a limited number of species. The main identified strains were Lactobacillus brevis, L. plantarum, L. casei, L. sakei, L. pentosus, L. farciminis and Leuconostoc mesenteroides.
Abstract: Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes. To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster. The PARP gene consisted of six translatable exons and spanned more than 50 kb. The DNA binding domain is encoded by exons 1-4. Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina. There are two cDNAs species in Drosophila. One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing. The expression of these two forms of PARP in E. coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive. On the other hand PARP I is active. A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly. These results suggest that the PARP gene plays an important function during the development of Drosophila.
Abstract: We here report an alternatively spliced form of PARP lacking exon 5 of the Drosophila PARP gene encoding the auto-modification domain. The alternative form of PARP (PARP II) consists 804 amino acids with a molecular weight of 92.3 kDa. The deduced amino acid sequence of PARP II was completely matched to that of PARP I encoded by a full-length Drosophila PARP cDNA, except it lacks the region corresponding to the auto-modification domain. To examine the function of PARP II, stable transformants of Rat-1 cells in which PARP II was ectopically expressed by MMTV-LTR were isolated and characterized. After induction with dexamethasone, PARP II transformants showed slower growth and showed morphological changes with loss of spindled shape compared to cells transformed with the vector or PARP I. The PARP II-transformed cells incorporated propidium iodide after induction; however, Annexin V and TUNEL analysis indicated these changes were not due to apoptosis.
Abstract: Caspase activities and two cDNA sequences have been identified in Drosophila melanogaster. To study the molecular events following the activation of the apoptotic pathway in D. melanogaster, S2 cells were treated with etoposide and the timing of the apoptotic events, such as caspase activation, mitochondrial pore opening, and loss of membrane asymmetry, was determined. Poly(ADP-ribose) polymerase (PARP) is known to be cleaved in the early phase of apoptosis in vertebrate systems. Little is known about the involvement of PARP cleavage in apoptosis in invertebrates. If PARP inactivation is a general event, this could mean that DNA repair enzymes need to be cleaved for the death pathway to be completed. We have found that in etoposide-treated cells, PARP protein is processed, but the nature of the cleavage is not known. Further experiments must be conducted and the peptide fragments must be sequenced to relate protease activities with PARP cleavage.
Abstract: To understand the biological function of poly(ADP-ribosyl)ation of proteins, we have isolated and characterized the gene for poly(ADP-ribose) polymerase from Drosophila melanogaster. Two approaches were taken to analyze the function of the poly(ADP-ribosyl)ation reaction. The first is analysis of the homology of the amino acid sequences of poly(ADP-ribose) polymerase from phylogenetically different eukaryotes, namely human, mouse, bovine, chicken, Xenopus laevis and Drosophila melanogaster and elucidation of the conserved amino acid sequences that appear to be important for the function of poly(ADP-ribose) polymerase. Analysis of the recombinant poly(ADP-ribose) polymerase which had truncated or mutated motifs expressed in E coli would confirm the importance of the conserved amino acid sequence. The interaction of poly(ADP-ribose) polymerase with other proteins involved in DNA repair, replication, recombination and transcription will clarify the function of poly(ADP-ribosyl)ation. The second approach is to get the mutants which have disruption in the poly(ADP-ribose) polymerase gene and to analyse the phenotypes of these mutants. The characterization of these mutants will be discussed.
Abstract: Steroid receptors and tamoxifen binding sites (TBS) were assayed in the soluble fraction of 121 primary breast cancers. Scatchard analysis of TBS in high speed supernatant (100,000 g) showed one population of binding sites; however, biphasic plots were obtained in low speed supernatants (40,000 g). Isoelectric focussing of supernatants preincubated with radioactive tamoxifen identified two classes of TBS (pI 4.1-4.6) which have different binding affinities and bind neither oestradiol nor diethylstilbestrol. Association between TBS and steroid receptors was: TBS positive/progesterone receptor positive 32.6%, TBS positive/glucocorticoid receptor positive 52.7%, TBS positive/oestrogen receptor positive 60% and TBS positive/androgen receptor positive 72.2%. We conclude that heterogeneous TBS are present in low speed fractions and can be easily separated from the oestrogen receptor by isoelectric focussing. The association between TBS and steroid receptor status could be of clinical value in the management of primary breast cancer.
Abstract: The activities of two DNA repair-related enzymes, poly(ADP-ribose) polymerase and DNA polymerase beta, and their mRNA levels were measured in 17 tissues of Wistar rats. A large variety in enzyme activity values could be detected in the tissues examined; the highest levels of activity for both enzymes were found in the testis. A good correlation between poly(ADP-ribose) polymerase activity and the level of the transcript of the gene coding for the enzyme was observed in many tissues. A less satisfactory correlation could be evidenced for DNA polymerase beta. The almost parallel amounts of the mRNAs for poly(ADP-ribose) polymerase and DNA polymerase beta in the tissues examined suggest a possible coexpression of the genes coding for these enzymes. Additional studies have been carried out in testis and liver by immunohistochemical techniques and by in situ hybridization analyses. While in the testis the spermatocytes were shown to contain both enzymes and their transcripts, in other types of cells this could not be observed. In the liver mRNAs and enzymes were only found in 20% of the hepatocytes. This may in part explain both the low levels of the mRNAs and the modest activities of the two enzymes in that tissue.
Abstract: The aim of this work was the testing of a diagnostic tool for the detection of allergenic proteins from stored product mites/cockroaches and house dust mites. Species-specific antibodies were previously tested for specificity and cross-reactivity, and detection limit by ELISA method. In this work, protein chips were manufactured either manually, using a MicroCaster, or robotically, using a SpotArray. Proteins used were six recombinant Kunitz-type protease inhibitors (PI), two of group A (with anti-cathepsin D and anti-trypsin activity), two of group B (with anti-trypsin and anti-chymotrypsin activity) and two of group C (with anti-cathepsin B activity), and the soybean Bowman-Birk inhibitor. Proteins were immobilized onto epoxy activated glass slides. Protein chips were hybridised with serial dilutions of extracts from adult mites/insects and from spent growth media (SGM), in order to capture the allergenic proteases. Protein A was labelled with Alexa-555 and used for detection of polyclonal antibodies bound on the chip surface. The detection limit was 0.5µg protein extract, for Aleuroglyphus ovatus and 0.75µg in Tyrophagus putrescentiae specimens and protein extracts from the faeces fraction (SGM). By comparison, the detection limit of ELISA method was 2.5µg. The protease inhibitor-chips can allow rapid screening of proteases and their specificity by analysing the binding to each class of inhibitors. Results of protease binding to each KPI and recognition of allergenic proteases showed the practicability of protein microarrays as a diagnostic tool for detection of mite/insect contaminants in foods and work environment.
Abstract: At CNR analyses of food products and compounds
with the use of innovative technologies were developed:
Identification: genes, proteins and metabolites in food products
and plants, biosynthesis pathways, biomarkers, DNA arrays and
expression profiling, Northern blots, RT-PCR
Analysis: effects of biocompounds on cell cultures, cell growth
and survival, biochemical assays, cell activation /inhibition,
Western blots, monitoring changes in cell phenotype, cell-to-cell
contact, analysis of gap-junctions using microinjection and
confocal microscopy
Detection: compounds / proteins in food using Protein arrays
and antibody chips (enzymes, allergens, protein-protein
interaction studies)
