Quantitation of the cytokines TNF-alpha, IL-8 and IL-10 in bovine milk using real-time TaqMan PCR.
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Animals↗
Biomedical subjects
P De Santis
Publications and source records attributed to P De Santis.
From the sequence to the superstructural properties of DNAs.
A theoretical model for predicting intrinsic and induced DNA superstructures as well as their thermodynamic properties is presented. Intrinsic sequence-dependent superstructures are evaluated by integrating local deviations from the canonical B-DNA of the different dinucleotide steps. Induced superstructures are obtained by adopting the principle of minimum deformation free energy, evaluated in the Fourier space, in the framework of first-order elasticity. Finally dinucleotide stacking energies and melting temperatures are considered to account for local flexibility. In fact the two scales are strongly correlated. The model works very satisfactorily in predicting the sequence-dependent effects on the DNA experimental behavior, such as the gel electrophoresis retardation, the writhe transitions in topologically constrained domains, the thermodynamic constants of circularization reactions as well as the nucleosome thermodynamic stability constants.
Algorithms↗
Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.
Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.
Animals↗
Mapping the intrinsic curvature and flexibility along the DNA chain.
The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a "natural" DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chain's intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.
DNA, Bacterial↗
Identification of protein domains on topological basis.
A theoretical method is proposed to identify structural domains in proteins of known structures. It is based on the distribution of the local axes of the polypeptide chain. In particular, a statistical analysis is applied to the contributions of the local axes to the absolute writhing number, a topological property of a space curve resulting from the number of self-crossings in the curve projections onto a unit sphere. This finding supports the hypothesis that topological requirements should be satisfied in the process of protein folding and in the final organization of the tertiary structures.
Databases, Factual↗
The main role of the sequence-dependent DNA elasticity in determining the free energy of nucleosome formation on telomeric DNAs.
Using a competitive reconstitution assay, we measured the free energy spent in nucleosome formation of eight telomeric DNAs, differing in sequence and/or in length. The obtained values are in satisfactorily good agreement with those derived from a theoretical model that allows the calculation of the free energy of nucleosome formation on the basis of sequence-dependent DNA elasticity, using a statistical thermodynamic approach. Both theoretical and experimental evaluations show that telomeres are characterized by the highest free energies of nucleosome formation among all the DNA sequences so far studied. The free energy of nucleosome formation varies according to the different telomeric sequences and the length of the fragments. Theoretical analysis and experimental mapping by lambda exonuclease show that telomeric nucleosomes occupy multiple positions spaced every telomeric repeat. Sequence-dependent DNA elasticity appears as the main determinant of the stability of telomeric nucleosomes and their multiple translational positioning.
Animals↗
A theoretical model for the prediction of sequence-dependent nucleosome thermodynamic stability.
A theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence is advanced. The model is based on a statistical mechanical approach, which allows the calculation of the canonical ensemble free energy involved in the competitive nucleosome reconstitution. It is based on the hypothesis that nucleosome stability mainly depends on the bending and twisting elastic energy to transform the DNA intrinsic superstructure into the nucleosomal structure. The ensemble average free energy is calculated starting from the intrinsic curvature, obtained by integrating the dinucleotide step deviations from the canonical B-DNA and expressed in terms of a Fourier series, in the framework of first-order elasticity. The sequence-dependent DNA flexibility is evaluated from the differential double helix thermodynamic stability. A large number of free-energy experimental data, obtained in different laboratories by competitive nucleosome reconstitution assays, are successfully compared to the theoretical results. They support the hypothesis that the stacking energies are the major factor in DNA rigidity and could be a measure of DNA stiffness. A dual role of DNA intrinsic curvature and flexibility emerges in the determination of nucleosome stability. The difference between the experimental and theoretical (elastic) nucleosome-reconstitution free energy for the whole pool of investigated DNAs suggests a significant role for the curvature-dependent DNA hydration and counterion interactions, which appear to destabilize nucleosomes in highly curved DNAs. This model represents an attempt to clarify the main features of the nucleosome thermodynamic stability in terms of physical-chemical parameters and suggests that in molecular systems with a large degree of complexity, the average molecular properties dominate over the local features, as in a statistical ensemble.
Base Sequence↗
Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results.
At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.
Animals↗
Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction.
Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.
Animals↗
Dual role of DNA intrinsic curvature and flexibility in determining nucleosome stability.
A statistical mechanistic approach to evaluate the sequence-dependent thermodynamic stability of nucleosomes is proposed. The model is based on the calculation of the DNA intrinsic curvature, obtained by integrating the nucleotide step deviations from the canonical B-DNA structure, and on the evaluation of the first order elastic distortion energy to reach the nucleosomal superstructure. Literature data on the free energy of nucleosome formation as obtained by competitive nucleosome reconstitution of a significant pool of different DNA sequences were compared with the theoretical results, and a satisfactorily good correlation was found. A striking result of the comparison is the emergence of two opposite roles of the DNA intrinsic curvature and flexibility in determining nucleosome stability. Finally, the obtained results suggest that the curvature-dependent DNA hydration should play a relevant role in the sequence-dependent nucleosome stability.
Base Sequence↗
Detection of Mycoplasma mycoides subspecies mycoides SC in clinical material by a rapid colorimetric PCR.
A colorimetric polymerase chain reaction (PCR) test for the detection of Mycoplasma mycoides subsp. mycoides SC (Mmm SC), the cause of contagious bovine pleuropneumonia (CBPP), was used to test clinical samples from suspect and known cases of the disease. A commercially available detection kit, pleuro TRAP from AMRAD, Australia, which captures amplified double stranded DNA in the wells of a microtitre plate was used. Tissues tested from animals ranging from a variety of anatomical locations included lung, lymph node, kidney, spleen, pleural fluid and nasal swabs. Polymerase chain reaction was conducted on DNA extracted from tissues as well as on culture-derived mycoplasmas. Positive results could be seen by the development of a yellow colour in microtitre wells after the enzymatic detection procedure indicating the presence of specifically amplified products. Spectrophotometric values of OD450for positive samples ranged from 0.211 to 1.409 compared to negative values of around 0.020 which reduced the subjectivity in the interpretation of results. Mmm SC was successfully identified from cultures of reference and field strains, nasal swabs and excised tissue by amplification with primers supplied in the kit. The system is suitable for automation and may be a cost effective alternative to gel electrophoresis for the screening of moderately large sample numbers as is the case in surveillance programmes.
Animals↗
DNA superstructural features and nucleosomal organization of the two centromeres of Kluyveromyces lactis chromosome 1 and Saccharomyces cerevisiae chromosome 6.
Superstructural features of the Kluyveromyces lactis chromosome 1 (KlCEN1) and of the Saccharomyces cerevisiae chromosome 6 (SCEN6) centromeric DNAs were evaluated using a theoretical method, developed by our group, and experimentally measured by gel electrophoretic retardation. Both methods show that, in spite of the remarkable AT richness of the two centromeric sequences, their curvature is not very high. However the peculiar sequence features of the two centromeres allow to organize highly stable nucleosomes, with a free energy about that of the nucleosome formed on the 5S RNA gene. The good agreement between experimental and theoretical evaluation of nucleosome free energies as well as of their multiple positioning shows that in centromeres both DNA curvature and flexibility are relevant in determining nucleosomal features.
Centromere↗
Multiple nucleosome positioning with unique rotational phasing on multimers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes: comparison between experimental and theoretical mapping.
Nucleosome positioning along two DNA tracts, corresponding to tetramers of the light-responsive elements of pea rbcS-3A and rbcS-3.6 genes, were studied by experimental (exonuclease III mapping and band shift electrophoresis) as well as theoretical methods. Multiple nucleosome positioning with unique rotational phase was derived from both methods in satisfactorily good agreement, if nucleosome dyad axis positions are considered. Theoretical and experimental distributions of nucleosome frequencies appear different, probably on account of DNA sequence dependent digestion kinetics of exonuclease III.
Base Sequence↗
Influence of DNA superstructural features and histones aminoterminal domains on mononucleosome and dinucleosome positioning.
Mononucleosome and dinucleosome positioning was studied in the complexes between two DNA fragments of different lengths, both containing a strongly curved sequence from Crithidia fasciculata kinetoplast, and histone octamers either normal or lacking aminoterminal domains. The results obtained by Exo III and DNase I selective digestion were: (a) The first and most stable nucleosome, formed with both types of histone octamers, is positioned on the curved sequence, showing a multiple dyad axis translational positioning with the same rotational phasing. This result is in very good agreement with the theoretical prediction, obtained by adopting a method developed by us, based on the evaluation of DNA distortion energy from the nucleotide sequence. (b) The second nucleosome has two main different positions. The first one, near the extremity of the DNA fragment opposite to the curved sequence, presents a higher frequency in the case of normal nucleosome, whereas an intermediate position appears populated with a higher frequency in the case of the "tailless' nucleosome.
Animals↗
A comparison of serological tests and gross lung pathology for detecting contagious bovine pleuropneumonia in two groups of Italian cattle.
Western and dot blotting techniques were compared with complement fixation tests (CFT), indirect enzyme-linked immunosorbent assays (ELISA), mycoplasma culture and gross lung pathology to detect Mycoplasma mycoides subspecies mycoides SC, the cause of contagious bovine pleuropneumonia (CBPP), in two groups of Italian cattle. None of the animals showed any clinical signs before slaughter. In group A, seven of the 20 cattle had characteristic lung lesions of acute and chronic CBPP but only six were positive by CFT. Western blotting detected antibody in eight of the animals, of which six had lesions and significant CFT titres (> 50 per cent fixation at a serum dilution of 1/10) and two had neither. In group B, seven of the 17 cattle had lesions characteristic of CBPP, and 12 were seropositive by CFT. Western blotting detected antibody in 13 of the animals including one which had a negative CFT titre. The ELISA was less sensitive than either CFT or Western blotting, detecting antibody in five animals in group A and nine animals in group B. The dot blotting test correlated well with Western blotting but gave a small number of ambiguous results. The causative organism was isolated from four of the 20 cattle in group A and six of the 17 cattle in group B.
Animals↗
Superstructural features of the upstream regulatory regions of two pea rbcS genes and nucleosomes positioning: theoretical prediction and experimental evaluation.
Nucleosome positioning on two 384 bp DNA fragments, obtained from the upstream regulatory region of two pea rbcS genes, relevant in photoregulated transcription, was predicted using our theoretical method, based on the evaluation of the sequence dependent DNA bending energy. The theoretical prediction was checked by experimental evaluation of nucleosome positions after in vitro reconstitution, by mapping Exonuclease III-resistant borders and by digesting monomeric sequences with various restriction enzymes. Both approaches satisfactorily confirmed the theoretical predictions, showing that the nucleotide sequence intrinsic bendability has a dominant role in nucleosome positioning.
Base Sequence↗
Influence of dynamic fluctuations on DNA curvature.
The effect of dynamic fluctuations on physical manifestations of DNA curvature such as electrophoretic retardation, circularization of DNA tracts and nucleosomes positioning is examined. It is shown that in all cases the main features of the processes can be satisfactorily explained by a static curvature model, which appears to be a good representation of time and ensemble averaged superstructures of DNA chains. The dynamic fluctuations around the average curvature appear to influence only the kinetics of these processes. In the case of polyacrylamide gel electrophoretic retardation it is demonstrated that the approximation of the static model holds on the assumption that dynamic fluctuations are independent from intrinsic curvature. The actual validity of the static model we proposed several years ago is satisfactorily demonstrated by the explanation and prediction of different experiments, such as cyclic permutation gel electrophoresis, differential DNAase I cleavage of cyclic versus linear DNA tracts and nucleosome positioning.
Animals↗
Different flexibility of the upstream regulatory regions of two differently expressed pea rbcS genes studied by theoretical evaluation of DNA distortion energy and cyclization kinetics.
Different superstructural features of the upstream regulatory regions of the two pea genes rbcS-3A and rbcS-E9 have been derived using a theoretical method, developed in our laboratory a few years ago, which evaluates the DNA distortion energy from a matrix of the deviations of the 16 possible dinucleotides from the standard B conformation. The theoretical analysis, which predicts different flexibilities of the regulatory regions of the two genes, is in satisfactorily good agreement with experimental evaluations from gel electrophoretic mobility and cyclization kinetics, suggesting a possible model to explain the largely different transcription efficiencies of the two genes.
Base Sequence↗