Binding by calmodulin is coupled to transient unfolding of the third FF domain of Prp40A.

Human pre-mRNA processing protein 40 homolog A (hPrp40A) is a splicing factor that interacts with the Huntington's disease protein huntingtin (Htt). Evidence has accumulated that both Htt and hPrp40A are modulated by the intracellular Ca2+ sensor calmodulin (CaM). Here we report characterization of the interaction of human CM with the third FF domain (FF3 ) of hPrp40A using calorimetric, fluorescence and structural approaches. Homology modeling, differential scanning calorimetry and small angle X-ray scattering (SAXS) data show FF3 forms a folded globular domain. CaM was found to bind FF3 in a Ca2+ -dependent manner with a 1:1 stoichiometry and a dissociation constant (Kd ) of 25 ± 3 µM at 25°C. NMR studies showed that both domains of CaM are engaged in binding and SAXS analysis of the FF3 -CaM complex revealed CaM occupies an extended configuration. Analysis of the FF3 sequence showed that the anchors for CaM binding must be buried in its hydrophobic core, suggesting that binding to CaM requires unfolding of FF3 . Trp anchors were proposed based on sequence analysis and confirmed by intrinsic Trp fluorescence of FF3 upon binding of CaM and substantial reductions in affinity for Trp-Ala FF3 mutants. The consensus model of the complex showed that binding to CaM binding occurs to an extended, non-globular state of the FF3 , consistent with coupling to transient unfolding of the domain. The implications of these results are discussed in the context of the complex interplay of Ca2+ signaling and Ca2+ sensor proteins in modulating Prp40A-Htt function.

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production.

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.

DNA typing methods for differentiation of yeasts related to dry-cured meat products.

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.

Characterization of molds from dry-cured meat products and their metabolites by micellar electrokinetic capillary electrophoresis and random amplified polymorphic DNA PCR.

Molds are common contaminants of dry-cured meat products in which mycotoxins could be synthesized if stored under favorable conditions. Thus, efficient and accurate characterization of the toxigenic molds from dry-cured meat products is necessary. A micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by 20 mold strains commonly found in dry-cured meat products. In addition, their random amplified polymorphic DNA (RAPD) genotypes were determined by using a PCR method. Although peak profiles of the secondary metabolites differed among mold strains of different species, they were similar in the same species. MECC analysis showed that 10 of the 20 molds tested produced mycotoxins, including patulin, penicillic acid, cyclopiazonic acid, mycophenolic acid, aflatoxin B1, sterigmatocystin, and griseofulvin. The RAPD analysis yielded a different pattern for each of the mold species tested. However, strains of the same species showed similar RAPD profiles. A high correlation between RAPD analysis and MECC was observed, since strains of the same species that showed similar RAPD patterns had similar profiles of secondary metabolites. RAPD patterns with primer GO2 and MECC profiles, either singly or combined, could be of great interest to distinguish toxigenic from nontoxigenic molds in dry-cured meat products.

Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.

A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.

Differentiation of Clostridium perfringens and Clostridium botulinum from non-toxigenic clostridia, isolated from prepared and frozen foods by PCR-DAN based methods.

During the elaboration process of prepared and frozen foods, Clostridium sp. have been reported. From those microorganisms, C. perfringens and C. botulinum may pose a high risk for the consumers. To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C. perfringens and C. boulinum should be established. In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C. perfringens from other Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characterization by API system. The restriction analysis of the 16S rDNA with Taq I and Rsa I showed at least the same sensitivity to differentiate C. perfringens from clostridial isolates as biochemical identification. However, the former method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6-8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.

Evaluation of microbial proteolysis in meat products by capillary electrophoresis.

AIMS: The available methods for evaluating proteolysis in meat products, particularly the contribution of micro-organisms, are expensive, time-consuming and require an unacceptable sample size. To minimize these problems, two capillary electrophoresis-based methods have been developed. METHODS AND RESULTS: Six Gram-positive, catalase-positive cocci, four moulds and three yeasts, isolated from dry-cured ham, were tested on sterile pork slices. Using the Capillary Gel Electrophoresis (CGE) method, changes in sarcoplasmic and myofibrillar proteins due to endogenous and microbial enzymes were detected. The Capillary Zone Electrophoresis (CZE) analysis allowed evaluation of bulk changes by micro-organisms in soluble nitrogen compounds. CONCLUSION: CGE analysis of myofibrillar proteins and CZE determination of soluble nitrogen compounds have proved to be valuable tools for evaluating proteolytic activity of endogenous and microbial origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The CGE and CZE methods developed can be used for a rapid and sensitive analysis of proteolysis in meat products.

Free amino acids and other non-volatile compounds formed during processing of Iberian ham.

Fifty-five legs from Iberian pigs were traditionally processed into dry cured hams. Free amino acids and other non-volatile compounds in the water-soluble fraction from the biceps femoris muscle were analyzed by HPLC. At the drying stage and in the last months in the cellar the largest increases in these water-soluble compounds took place. There was a clear influence on free amino acid formation of salt content and on the formation of peptides of the temperature at each processing stage. As the amount of non-volatile compounds in the water-soluble fraction increases with processing time, their determination could provide a maturation index for Iberian ham.

Rapid differentiation of Staphylococcus aureus from staphylococcal species by arbitrarily primed-polymerase chain reaction.

An arbitrarily primed-polymerase chain reaction (AP-PCR) method was optimized to differentiate Staphylococcus aureus from other staphylococcal species, using DNA from crude cell extract. From the different assays carried out, the best resolution of the band patterns was obtained when the reaction mixture contained 200 micromol l(-1) dNTPs, 200 ng primer, 1 U Taq DNA polymerase and 3 mmol l(-1) MgCl2 and the amplification conditions were: initial denaturation of 94 degrees C for 1 min, primer annealing of 30 degrees C for 1.5 min, DNA extension at 55 degrees C for 5 min and final extension at 55 degrees C for 5 min. The results of the characterization of the staphylococcal isolates by AP-PCR are in accordance with those of the biochemical identification by the API Staph System, time of analysis of the AP-PCR being only 6-7 h. Thus, this technique could be a useful method for microbial quality assurance.

Microbial populations and volatile compounds in the 'bone taint' spoilage of dry cured ham.

'Bone taint' is one of the most important causes of spoilage of dry cured ham. This alteration is characterized by a foul-smelling odour. The microbial population and volatile compounds associated with incipient 'bone taint' were evaluated. Enterobacteriaceae species were found at levels of 7.94 x 10(5) cfu g(-1) in spoiled hams and were not detected in unspoiled hams. Serratia sp. and Proteus sp. are the main organisms characterized. The volatile compounds from the spoiled hams give rise to higher levels of ketones, alcohols and esters than unspoiled hams, that could be originated by microbial metabolism of the above bacteria. Thus, volatile and Enterobacteriaceae analyses should be used to determine this incipient spoilage in the meat industry.

Changes in intramuscular lipids during ripening of Iberian dry-cured ham.

Thirty-one thighs were obtained from Iberian pigs fattened with acorns and were processed during 22 months in the traditional dry-curing process. Lipolysis affecting intramuscular fat during the processing of Iberian dry-cured ham has been analyzed by studying the changes of glycerides, phospholipids and free fatty acids in lipids from Biceps femoris muscle. Little change affected the fatty acid composition of glycerides during processing. A double-phased increase in the acidity values and a decrease in the quantity of fatty acids of phospholipids during the processing were observed. There seems to be a relationship between the extension of the lipolysis taking place during the maturing and the processing conditions and raw material used.

Evaluation of proteolytic activity of micro-organisms isolated from dry cured ham.

In order to determine the possible contribution of micro-organisms to the ripening of meat products, 48 cocci, 18 moulds and 20 yeasts isolated from dry-cured Iberian ham were evaluated for proteolytic activity. Two specific methods were used: the ability to hydrolyse myosin in broth and, for those strains showing high activities, hydrolysis on both myofibrillar and sarcoplasmic proteins on pork slices. Moulds and cocci showed the highest proteolytic activity for myosin in broth. Both myofibrillar and sarcoplasmic proteins were recovered at lower rates from inoculated than from sterile incubated pork. The deepest changes in myofibrillar and sarcoplasmic proteins were originated by one strain each of Penicillium chrysogenum and Staphylococcus xylosus, respectively. Only small changes were observed in the concentrations of free amino acids from inoculated pork slices, except for the samples with P. chrysogenum, where there were increases in all free amino acids. Thus, P. chrysogenum makes a significant contribution to proteolysis during the ripening of dry-cured meat products.

Evaluation of microbial hazards during processing of Spanish prepared Flamenquín.

Flamenquín is a traditional, prepared, frozen meat product from the south of Spain made with minced pork, chicken, and cooked ham. Since it is a prepared raw meat product some microbial hazards could be associated with the process of making it. Microbiological analyses have been performed throughout the various steps of processing over a 1-year period to evaluate microbial hazards in the commercial process. High levels of microorganisms were observed all through the processing of this product, the mincing and mixing steps being where major microbial contamination was observed. Pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli and Pseudomonas aeruginosa were detected during processing. Raw materials and food handlers were the principal sources of microbial contamination. A modification of processing to include a heating step after mincing and mixing and an improvement in hygiene practices could eliminate the microbial hazards. Both modifications should be noted for the implementation of a hazard analysis of critical control points (HACCP) program in commercial flamenquín processing.

Effects of salt and temperature on proteolysis during ripening of Iberian ham.

Fifty-five hams from Iberian pigs were processed using two different dry-curing techniques, traditional and modern. Salt content, non-protein nitrogen and its fractions (peptide, amino acid and volatile basic nitrogen) from Biceps femoris muscles were quantified. The existence of an overlapping effect of both temperature and salt content on the general non-protein nitrogen production was observed. The most intense proteolytic breakdown took place when higher temperatures were reached during the drying stage. The difference in salt concentration seems to contribute to generating different quantities in the non-protein nitrogen fractions. The inclusion at the end of the cellar stage of a stuffing period would permit increasing the accumulation of free amino acid in high salted hams.

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe.

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2.1-8.1 cfu ml-1) for types A and B, but rather low (10(4) cfu ml-1) for types E and F. However, after enrichment at 37 degrees C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g-1 of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.

Composition and toxigenic potential of the mould population on dry-cured Iberian ham.

The fungal population on dry-cured Iberian ham can be essential to the development of the product's unique characteristics, but health hazards due to mycotoxins may be significant. We examined the natural fungal population of Iberian hams during ripening at three different locations. Chloroform extracts from 59 selected isolates were tested for toxicity to brine shrimp larvae and VERO cells, for mutagenicity in the Ames test and for antimicrobial activity against Staphylococcus aureus. The diversity of moulds increased during ripening. Penicillium commune, Penicillium chrysogenum, Penicillium aurantiogriseum, Penicillium expansum and Penicillium echinulatum dominated most of the ripening time; however, the Eurotium species, particularly E. herbariorum and E. repens, increased in the final product. Using the above tests, most moulds were toxigenic. The toxigenic potential of the fungal population increased as the processing progressed. To minimize health hazards from uncontrolled fungal populations, we identified non toxigenic strains of Penicillium chrysogenum that could be used as starters in dry-cured hams.

Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.

Yeast population during ripening of dry-cured Iberian ham.

The relationship between the superficial yeast population and the ripening conditions of Iberian dry cured hams has been studied for three different locations. Tentative identifications were carried out for 836 isolates. Candida zeylanoides was the dominating yeast in early stages, whereas more than 99% of isolates from the surface of matured hams were identified as Debaryomyces hansenii. A great diversity of strains of C. zeylanoides and D. hansenii was found. The characteristic pattern of isolates from the various locations and the selection of various strains of D. hansenii during ripening make the study of the yeasts useful for estimating the progress of maturation.

Characterization of Staphylococcus spp. and Micrococcus spp. isolated from Iberian ham throughout the ripening process.

The Iberian dry cured ham is an uncooked meat product highly appreciated because of its characteristic flavour. This product is obtained from highly marbled Iberian pig hindlegs after 18-24 months of maturation under natural environmental conditions. The role of Micrococcaceae in the development of the aroma characteristics of this products remains unclear. Identification of Gram-positive, catalase-positive cocci isolated from Mannitol Salt Agar plates showed that Staphylococcus xylosus followed by Staphylococcus equorum are the predominant organisms, even after 16 months of maturing. A remarkable variety of types of both staphylococci and micrococci are detected at any sampling time. The metabolic activities of these organisms could contribute to the characteristics of the final product.