A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Comparative cytogenetic analysis of the “Sylvaemus” group of Apodemus (Rodentia, Muridae): A. sylvaticus from Sicily and A. flavicollis from the central Apennines

Chromosome mapping of three common markers, ie major and 5S rRNA genes (rDNA) and telomeric repeats, and conventional chromosome bandings were applied to two sibling species, Apodemus sylvaticus Linnaeus, 1758 and A. flavicollis Melchior, 1834, to further investigate intra- and interspecific karyological differentiation in the genus Apodemus. A slight variation of the rDNA-patterns was detected between the two Apodemus species. In both of them, the major NORs were located on autosome pairs 8, 11, 12 and 22, while the two other rDNA sites detected on chromosomes 7 and 21 were variable in, respectively, A. sylvaticus and A. flavicollis. Several tiny rDNA sites were present on the sex chromosomes in both species, but their incidence was lower in A. flavicollis. Single 5S rDNA chromosomal sites were conserved on chromosome pair 20. No interstitial sites of telomeric repeats were present in either species. In the Sicilian population of A. sylvaticus, the constitutive heterochromatin pattern corresponded to the “sylvaticus-E1” cytotype, while A. flavicollis had a species-specific pattern restricted to centromeres of all chromosomes. The results are discussed in relation to cytogenetic data available for the genus, with emphasis on the Sylvaemus group/subgenus.     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Functional,radiological and biological markers of alveolitis and infections of the lower respiratory tract in patients with systemic sclerosis

Background

A progressive lung disease and a worse survival have been observed in patients with systemic sclerosis and alveolitis. The objective of this study was to define the functional, radiological and biological markers of alveolitis in SSc patients.

Methods

100 SSc patients (76 with limited and 24 with diffuse disease) underwent a multistep assessment of cardiopulmonary system: pulmonary function tests (PFTs) every 6–12 months, echocardiography, high resolution computed tomography (HRCT) and bronchoalveolar lavage (BAL), if clinically advisable. Alveolar and interstitial scores on HRCT and IL-6 plasma levels were also assessed as lung disease activity indices.

Results

90 SSc patients with abnormal PFTs and 3 with signs and/or symptoms of lung involvement and normal PFTs underwent HRCT and echocardiography. HRCT revealed evidence of fibrosis in 87 (93.5%) patients, with 55 (59.1%) showing both ground glass attenuation and fibrosis. In 42 patients who had exhibited ground glass on HRCT and consented to undergo BAL, 16 (38.1%) revealed alveolitis. 12 (75%) of these patients had restrictive lung disease (p < 0.0001) and presented diffuse skin involvement (p = 0.0009). IL-6 plasma levels were higher in patients with alveolitis than in patients without (p = 0.041). On logistic regression model the best independent predictors of alveolitis were diffuse skin involvement (OR(95%CIs):12.80(2.54–64.37)) and skin score > 14 (OR(95%CIs):7.03(1.40–34.33)). The alveolar score showed a significant correlation with IL-6 plasma levels (r = 0.36, p = 0.001) and with the skin score (r = 0.33, p = 0.001). Cultures of BAL fluid resulted positive in 10 (23.8%) of the 42 patients that underwent BAL and after one year a deterioration in PFTs occurred in 8 (80%) of these patients (p = 0.01). Pulmonary artery systolic pressure ≥ 40 mmHg was found in 6 (37.5%) patients with alveolitis.

Conclusion

We found alveolitis only in 38.1% of the patients who had exhibited ground glass on HRCT and then underwent BAL, probably because the concomitant fibrosis influenced results. A diffuse skin involvement and a restrictive pattern on PFTs together with ground glass on HRCT were judged possible markers of alveolitis, a BAL examination being indicated as the next step. Nevertheless BAL would be necessary to detect any infections of the lower respiratory tract that may cause further deterioration in lung function.     

Astressin-amide and astressin-acid are structurally different in dimethylsulfoxide

The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an alpha-helical conformation, with a small kink around Gln(26). However, astressin-amide has a well-defined helical structure from Leu(27) to Ile(41) and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858-4863). In contrast, astressin-acid has an irregular helical conformation from Arg(35) onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation.     

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

It has recently been shown that the biological activity of the second generation kinin B1 receptor selective antagonist, desArg10 HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg10 HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide DTic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg10 HOE 140 to that of an agonist.     

Essential role for ADAM19 in cardiovascular morphogenesis

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.     

HYBRIDIZATION,NATURAL SELECTION,AND EVOLUTION OF REPRODUCTIVE ISOLATION: A 25‐YEARS SURVEY OF AN ARTIFICIAL SYMPATRIC AREA BETWEEN TWO MOSQUITO SIBLING SPECIES OF THE Aedes mariae COMPLEX

Natural selection can act against maladaptive hybridization between co‐occurring divergent populations leading to evolution of reproductive isolation among them. A critical unanswered question about this process that provides a basis for the theory of speciation by reinforcement, is whether natural selection can cause hybridization rates to evolve to zero. Here, we investigated this issue in two sibling mosquitoes species, Aedes mariae and Aedes zammitii, that show postmating reproductive isolation (F1 males sterile) and partial premating isolation (different height of mating swarms) that could be reinforced by natural selection against hybridization. In 1986, we created an artificial sympatric area between the two species and sampled about 20,000 individuals over the following 25 years. Between 1986 and 2011, the composition of mating swarms and the hybridization rate between the two species were investigated across time in the sympatric area. Our results showed that A. mariae and A. zammitii have not completed reproductive isolation since their first contact in the artificial sympatric area. We have discussed the relative role of factors such as time of contact, gene flow, strength of natural selection, and biological mechanisms causing prezygotic isolation to explain the observed results.     

Rapid Annotation of Anonymous Sequences from Genome Projects Using Semantic Similarities and a Weighting Scheme in Gene Ontology

Background

Large-scale sequencing projects have now become routine lab practice and this has led to the development of a new generation of tools involving function prediction methods, bringing the latter back to the fore. The advent of Gene Ontology, with its structured vocabulary and paradigm, has provided computational biologists with an appropriate means for this task.

Methodology

We present here a novel method called ARGOT (Annotation Retrieval of Gene Ontology Terms) that is able to process quickly thousands of sequences for functional inference. The tool exploits for the first time an integrated approach which combines clustering of GO terms, based on their semantic similarities, with a weighting scheme which assesses retrieved hits sharing a certain number of biological features with the sequence to be annotated. These hits may be obtained by different methods and in this work we have based ARGOT processing on BLAST results.

Conclusions

The extensive benchmark involved 10,000 protein sequences, the complete S. cerevisiae genome and a small subset of proteins for purposes of comparison with other available tools. The algorithm was proven to outperform existing methods and to be suitable for function prediction of single proteins due to its high degree of sensitivity, specificity and coverage.