High pressure induced changes on sarcoplasmic protein fraction and quality indicators

The combined effect of pressure and mild temperature treatments on bovine sarcoplasmic proteins and quality parameters was assessed. M. longissimus dorsi samples were pressurised in a range of 200–600 MPa and 10–30 °C. High Pressure Processing (HPP) induced a reduction of protein solubility (p < 0.001) compared to non-treated controls (NT), more pronounced above 200 MPa. HPP at pressures higher than 200 MPa induced a strong modification (p < 0.001) of meat colour and a reduction of water holding capacity (WHC). SDS–PAGE analysis demonstrated that HPP significantly modified the composition of the sarcoplasmic protein fraction. The pressurisation temperature mainly affected protein solubility and colour; a smaller effect was observed on protein profiles. Significant correlations (p < 0.001) between sarcoplasmic protein solubility and both expressible moisture (r = −0.78) and colour parameters (r = −0.81 to −0.91) suggest that pressure induced denaturation of sarcoplasmic proteins could influence to some extent WHC and colour modifications of beef. Changes in protein band intensities were also significantly correlated with protein solubility, meat lightness and expressible moisture. These results describe the changes induced by HPP on sarcoplasmic proteins and confirm a relationship between modification of the sarcoplasmic protein fraction and alteration of meat quality characteristics

Bioequivalence of meat products treated with high pressure processing

The effects of high pressure on the composition of food products have not been evaluated extensively. Since, it is necessary to take in consideration the possible effects in basis to the changes induced in the bio molecules by the application of high pressures. The main effect on protein is the denaturation, because the covalent bonds are not affected; however hydrogen bonding, hydrophobic and intermolecular interactions are modified or destroyed. 1 High pressure can modify the activity of some enzymes. If this is done the proteolysis and lipolysis could be more or less intense and the content of free amino acids and fatty acids will be different. This could be related to the bioavailability of these compounds. Low pressures (100 MPa) have been shown to activate some enzymes (monomeric enzymes). Higher pressures induce loss of the enzyme activity. However some enzymes are very stable (ex. Lipase ~ 600 - 1000 MPa). Lipoxygenase is less stable, and there is little information about the effects on antioxidant enzymes. Other important issue is the influence of high pressure on oxidation susceptibility. This could modify the composition of lipids if the degree of the oxidation would have been higher or lower than in the traditional product. Pressure produces the damage of cell membranes favouring the contact between substrates and enzymes, exposure to oxidation of membrane fatty acids and loos of the efficiency of vitamin E. These effects can also affect to protein oxidation. In this study different compounds were analysed to establish the differences between non-treated and high-pressure treated products.

Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis

The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid (OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as model species. Shellfish matrix loading limits for the PPIA have been established, according to the shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48). Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs. The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Tandem chimeric transcription as a means to increase protein diversity in the human genome

The “one-gene, one-protein” rule, coined by Beadle and Tatum, has been fundamental to molecular biology. The rule implies that the genetic complexity of an organism depends essentially on its gene number. The discovery, however, that alternative gene splicing and transcription are widespread phenomena dramatically altered our understanding of the genetic complexity of higher eukaryotic organisms; in these, a limited number of genes may potentially encode a much larger number of proteins. Here we investigate yet another phenomenon that may contribute to generate additional protein diversity. Indeed, by relying on both computational and experimental analysis, we estimate that at least 4%–5% of the tandem gene pairs in the human genome can be eventually transcribed into a single RNA sequence encoding a putative chimeric protein. While the functional significance of most of these chimeric transcripts remains to be determined, we provide strong evidence that this phenomenon does not correspond to mere technical artifacts and that it is a common mechanism with the potential of generating hundreds of additional proteins in the human genome.

Estimation of protein coding density in a corpus of DNA sequence data

A number of experimental methods have been reported for estimating the number of genes in a genome, or the closely related coding density of a genome, defined as the fraction of base pairs in codons. Recently, DNA sequence data representative of the genome as a whole have become available for several organisms, making the problem of estimating coding density amenable to sequence analytic methods. Estimates of coding density for a single genome vary widely, so that methods with characterized error bounds have become increasingly desirable. We present a method to estimate the protein coding density in a corpus of DNA sequence data, in which a ‘coding statistic’ is calculated for a large number of windows of the sequence under study, and the distribution of the statistic is decomposed into two normal distributions, assumed to be the distributions of the coding statistic in the coding and noncoding fractions of the sequence windows. The accuracy of the method is evaluated using known data and application is made to the yeast chromosome III sequence and to C.elegans cosmid sequences. It can also be applied to fragmentary data, for example a collection of short sequences determined in the course of STS mapping.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

Identifying protein-coding genes in genomic sequences

The vast majority of the biology of a newly sequenced genome is inferred from the set of encoded proteins. Predicting this set is therefore invariably the first step after the completion of the genome DNA sequence. Here we review the main computational pipelines used to generate the human reference protein-coding gene sets.