[An atypical form of neurosarcoidosis]. / Neurosarcoïdose de présentation atypique.

INTRODUCTION: Nervous system involvement occurs in 5 to 15% of the patients with sarcoidosis. Neurosarcoidosis remains very difficult to diagnose because clinical presentation and imaging characteristics lack specificity. OBSERVATION: We report a 26-year-old man who gradually developed headaches, memory disturbance and epilepsy. CT-scan and MRI showed a temporal-parietal cystic mass, secondary to a rare and focal form of hydrocephalus, called "trapped temporal horn" revealing neurosarcoidosis. CONCLUSION: The "entrapped temporal horn" is due to an obstacle on the cerebrospinal fluid pathway at the trigone of the lateral ventricle that seals off the temporal horn and the choroid plexus from the rest of the ventricular system. The obstacle is related to the granulomatous tissue of sarcoidosis. Therefore, the "trapped temporal horn" acts as a space occupying process, causing headaches, memory pain, hemiparesis, homonymous hemianopsia, and requires medico-surgical management.

Carcase characteristics and qualitative meat traits of three Italian local chicken breeds.

1. An experiment involving 60 male chickens reared in an organic production system was carried out in order to investigate carcase characteristics and qualitative meat traits of three slow-growing Italian local breeds of chicken (Ermellinata, Padovana and Pepoi). 2. Chicks were randomly selected at hatch, raised together under the same conditions, slaughtered at 190 d of age, dissected for carcase traits, and meat (breast and thigh) stored for subsequent analysis of quality parameters. 3. Ermellinata (EA) chickens were significantly different from Padovana (PA) and Pepoi (PI) chickens for live, carcase and thigh weights. Breeds were also different for breast muscle protein content (EA > PI and PA), shear force (PA < EA and PI) and cooking loss (PI > PA and EA) values. 4. The CIE system values of lightness (L*), redness (a*), and yellowness (b*) evidenced a distinctive darker meat and lighter skin colour of PA breast meat. 5. Polyunsaturated fatty acids composition of breast meat was similar among the analysed breeds. EA had significantly higher saturated but significantly lower monounsaturated fatty acid contents than the other two breeds.

Genetic characterization of local Italian breeds of chickens undergoing in situ conservation.

The objectives of this study were to determine genetic variation and to analyze population structure of 6 Italian local chicken breeds involved in a conservation program. Twenty microsatellite markers were investigated in 337 birds belonging to 6 breeds: Ermellinata di Rovigo, Robusta Maculata, Robusta Lionata, Pépoi, Padovana, and Polverara; a commercial layer cross was used as reference. One hundred twelve alleles were detected in the overall population, with a mean number of 5.6 +/- 2.1 alleles per locus. For the local breeds, the observed and expected heterozygosity ranged from a minimum of 0.240 to a maximum of 0.413 and from 0.243 to 0.463 for the Pépoi and Polverara breeds, respectively. Deviation from Hardy-Weinberg equilibrium was observed in 5 breeds and in the commercial cross. The overall population heterozygote deficiency was 0.427, the average inbreeding coefficient was 0.097, and the heterozygote deficiency due to breed subdivisions was 0.437. Reynolds' distances were used to draw an unrooted neighbor-joining tree, which topology gave information on the genetic origin of these breeds and confirmed their known history. The estimated molecular kinship within a breed ranged from 0.559 to 0.769, evidencing high coancestry. Structure analysis was performed to detect the presence of population substructures. Inferred clusters corresponded to the different breeds, without presence of admixture. The exception was the Polverara breed, for which a more complex genetic structure was found. The results supported the decision of safeguarding these breeds as an important reservoir of genetic diversity and confirmed the usefulness of microsatellite markers to characterize and to monitor genetic variability in local chicken breeds.

Genomic DNA fingerprinting of indigenous chicken breeds with molecular markers designed on interspersed repeats.

In Italy more than fifty different local breeds of chicken (Gallus gallus L.) are known to have been present in the past. The overall situation is now critical since most of these breeds are becoming extinct or threatened and only a few are subject of conservation plans. The use of molecular markers for the analysis of chicken populations could help in characterizing their genetic variation and preserving them from genetic erosion. valuable and irreplaceable sources of chicken germplasm from indigenous populations of the veneto region were analyzed by means of DNA fingerprinting with molecular markers designed on interspersed mini- and microsatellite repeats. The identification of either among-breed discriminant or breed-specific markers was based on the S-SAP and M-AFLP systems derived from the AFLP technology. Genomic DNA fingerprints were generated in 84 individuals belonging to six local breeds (Ermellinata, Padovana, Pépoi, Polverara, Robusta Lionata and Robusta Maculata) and one commercial line used as reference standard. A number of variation statistics were computed to assess the genetic variability within and relatedness among breeds: the effective number of alleles per locus (n(e)= 1.570), total and single-breed genetic diversity (H(T)= 0.366 and H(S)= 0.209, respectively) and the fixation index (G(ST)= 0.429). The mean genetic similarity coefficients within and between local breeds were 0.769 and 0.628, respectively. Markers useful for the genetic traceability of breeds revealed significant sequence similarities with either genic or intergenic regions of known chromosome position. Sequence tagged site primers were designed for the most discriminant markers in order to develop multiplex non-radioactive genomic PCR assays. Analysis of the population structure along with individual assignment tests successfully identified all breed clusters and subclusters. The vast majority of animals were correctly allocated to their breed of origin, demonstrating the suitability and reliability of the chosen AFLP-derived marker systems for detecting population structure and tracing individual breeds. The local breeds have been preliminarily identified according to sequence-specific SNPs and haplotypes and the polymorphism information content of genomic AFLP-derived markers is reported and critically discussed.

Genetic variation and population structure of Italian native sheep breeds undergoing in situ conservation.

The genetic variability and presence of population substructures in 4 native Northern Italian sheep breeds, Alpagota, Brogna, Foza, and Lamon, undergoing in situ conservation, and 1 widespread Italian breed, Bergamasca, were studied by investigating 19 microsatellite markers. The breeds showed considerable genetic variability in terms of number of alleles and heterozygosity, with the exception of Alpagota, which was the least variable (0.607). Nevertheless, a significant deficit of heterozygotes was observed in each breed due to rather increased levels of inbreeding or to the presence of population substructures, probably caused by increased genetic variation in the founder populations. The analyses evidenced clear genetic differentiation (F(ST) = 0.085), reduced levels of admixture, and presence of private alleles among the breeds, confirming their genetic uniqueness. In particular, according to Reynolds genetic distances, Alpagota was the most differentiated, perhaps because it had been bred mostly in a rather isolated area. Loss of any of the investigated breeds would result in a loss of genetic diversity ranging from 0.5 to 1.6% of the total observed gene diversity. Results supported the decision to safeguard these breeds as important reservoirs of genetic diversity and suggested breeding and mating practices to maintain variability and to overcome within-breed substructures.

Effect of Holstein Friesian and Brown Swiss breeds on quality of milk and cheese.

In Italy, more than 75% of milk is used for cheese making. For this reason, milk composition and coagulation traits and cheese quality represent the most important tools for the economic development of the dairy sector. In particular, cheese quality varies in relation to cheese-making technology and breed of cow. The aim of this study was to investigate the effect of 3 types of milk, originating from Holstein-Friesian (HF), Brown Swiss (BS), and mixed of both breeds, on vat milk characteristics, cheese yield, and quality in 3 different typical Italian cheese-making conditions (Casolet, Vezzena, and Grana Trentino). One hundred forty-four cows (66 HF and 78 BS) were involved, and a total of 24 vats of milk were evaluated. At maturity, 30, 21, and 16 wheels of Casolet, Vezzena, and Grana Trentino cheese were analyzed. Brown Swiss cows yielded 9% less milk per day than HF cows, but milk showed greater contents of protein, casein, titratable acidity, and better rennet coagulation time and curd firmness than HF milk. The chemical composition and cholesterol content of the 3 types of cheese were similar between breeds, whereas the cheese made with BS milk showed greater contents of monounsaturated and polyunsaturated fatty acids. Cheese made with BS milk had greater b* (yellow component) than HF. Cheese yield, recorded at different ripening times, demonstrated that BS milk yielded more cheese than HF. Mixed milk showed values, on average, intermediate to HF and BS milk characteristics, and this trend was confirmed in cheese yield at different ripening times.

Genetic characterization of the Burlina cattle breed using microsatellites markers.

The present study was a contribution on the genetic characterization of the Burlina local cattle breed, and an approach to understanding the relationships between Burlina, Holstein Friesian and Brown Swiss which represent the majority of the dairy cattle reared nowadays in North-East Italy. The obtained results helped to clarify the genetic diversity and distinctiveness of Burlina population. In particular, the low genetic distance between Burlina and Holstein Friesian and the assignment of a moderate percentage of Burlina animals to Holstein Friesian suggested that crosses between them took place in the past, while crosses with Brown Swiss seemed to be less frequent. However, analyses of marker genotypes, showed a cluster with only Burlina individuals, which demonstrates the genetic distinctness of this breed. The Burlina breed showed the highest variability among the analysed breeds and its inbreeding coefficient was low. The data contribute to the feasibility of a conservation and selection programme for this breed and the results are useful for the implementation of a conservation strategy that should aim to conserve animals where the contribution from foreign breeds is as small as possible.

Breed assignment test in four Italian beef cattle breeds.

The assessment of a method able to assign individuals to the breed of origin is needed to certify origin and quality of livestock products. A set of 21 microsatellites was tested for breed identification in four native Italian beef breeds: Chianina, Marchigiana, Romagnola, and Piemontese. Two statistical approaches, based on maximum likelihood and on a Bayesian method, were evaluated. Different marker sets, chosen in order of the highest gene diversity and F(ST) estimates were also tested. The Bayesian method performed better, achieving a correct assignment rate of about 90% even with six microsatellites. The marker sets with the highest gene diversity were shown to perform best. Considering a threshold probability of 90%, only 52.5% of the genotypes were correctly allocated. Such results are mainly due to the low genetic differentiation estimates among breeds (F(ST)=0.049). These findings suggest that markers with high gene diversity and the presence of private alleles should be investigated and the Bayesian method used.

Heritabilities and genetic correlations of body condition score and calving interval with yield, somatic cell score, and linear type traits in Brown Swiss cattle.

This study aimed to estimate genetic parameters for body condition score (BCS), calving interval (CI), somatic cell score (SCS), yield, and linear type traits for the Italian Brown Swiss cattle population. A total of 32,359 records of first-parity lactating cows were collected from 2002 to 2004 in 4,885 dairy herds. The pedigree file included 96,661 animals. Multiple-trait animal models were analyzed using REML to estimate (co)variance components without repeated observations on traits. The estimated heritability was 0.15 for BCS, 0.05 for CI, and 0.06 for SCS, and ranged from 0.09 to 0.14 for test-day yield traits and from 0.07 to 0.32 for linear type traits. The genetic correlations of CI with yield and most linear type traits were positive, whereas the correlation between CI and BCS was negative (-0.35). For type traits, BCS showed, in general, a moderately negative genetic correlation except for strength, pastern, and heel height. The genetic correlation of CI or BCS with SCS was moderately low but favorable (0.19 and -0.26, respectively). The estimated correlations indicated that selection for greater yield and type traits can exert unfavorable effects on the reproductive ability of cows. To counterbalance these effects and to carry out early prediction of breeding values of bulls for fertility, inclusion of BCS in the breeding program is advisable.

Genetic traceability of livestock products: A review.

Traceability is the ability to maintain the identification of animal, or animal products, all along the production chain. It represents an essential tool to safeguard public and animal health and to valorize typical production systems. European food legislation is particularly strict and traceability systems, based on product labeling, have become mandatory in all European countries. However, the implementation of this system does not ensure consumers against fraud. Paper documents can be counterfeit so researchers have focused on the study of genetic traceability systems based on products identification through DNA analysis. In fact DNA is inalterable, detectable in every cell, resistant to heat treatments, and allows for individual, breed or species identification. Even if results are promising, these techniques are too expensive to be converted in routine tests but they could be a trusted tool for verification of suspected fraud. The present review proposes a synthesis of the major advances made in individual, breed, and species genetic identification in the last years, focusing on advantages and disadvantages and on their real future applications for animal productions.

Assessing genetic diversity in indigenous Veneto chicken breeds using AFLP markers.

Genetic variation in four indigenous chicken breeds from the Veneto region of Italy was assessed using amplified fragment length polymorphism (AFLP) markers. A total of 99 individuals were analysed using three AFLP primer combinations that produced 70 polymorphisms. Four indigenous Veneto chicken breeds (Ermellinata, Padovana, Pépoi and Robusta) and a reference broiler line were included in the analysis. Breed-specific markers were identified in each breed. The expected heterozygosity did not differ significantly among the indigenous Veneto chicken breeds and the broiler line. The coefficient of gene variation (Gst) value across loci indicated that almost half of the total variability was observed among breeds. Nei's standard genetic distance between pairs of breeds showed that the distance between the broiler line and the Pépoi breed was greater than the distances between the broiler line and the other three chicken breeds. Cluster analysis based on standard genetic distances between breeds indicated that the Padovana and Pépoi breeds were closely related. Factorial analysis based on a binary matrix of the AFLP data showed a clear distinction of all breeds.

NMR studies of protein surface accessibility.

Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.

Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe.

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.

WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability.

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.

Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water.

A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H2O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.

Use of organic solvents and small molecules for locating binding sites on proteins in solutions.

Application of a modified ePHOGSY and other NMR experiments to an H2O-DMSO solution of the protein FKBP12 identified the presence of one molecule of DMSO bound in the substrate binding site. It occupies the same spatial region occupied by the pipecolidine moiety of the immunosuppressive drugs FK506 and Rapamycin complexed to the protein. The binding constant K(D) for ths DMSO molecule was only 275 mM. A substructure search of small molecules similar to DMSO resulted in the identification of molecules with improved binding affinity. This work represents a clear example of the powerful interplay of molecular modelling and NMR.

Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins.

A novel variant of the 13C/15N ω2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.

Heteronuclear X-filter 1H PFG double-quantum experiments for the proton resonance assignment of a ligand bound to a protein.

A novel X-filter experiment based on 1H PFG DQ spectroscopy is described. Excellent suppression of proton bound to 13C and 15N is achieved. Successful application of the method to a protein-ligand complex is demonstrated.

ePHOGSY experiments on a paramagnetic protein: location of the catalytic water molecule in the heme crevice of the oxidized form of horse heart cytochrome c.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by 1H NMR spectroscopy. Application of ePHOGSY (enhanced protein hydration observed through gradient spectroscopy) experiments over a paramagnetic molecule provided firm spectroscopic evidence of the presence of a water molecule in the heme crevice. A few intermolecular NOEs have been used to locate the water molecule at about 0.65 nm away from the iron atom and to compare the position observed in solution with that observed in the crystal structure and in solution for the reduced state. The resulting picture is that there is a detectable movement of the water molecule upon oxidation.