Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).     

Pre-auricular tags and associated anomalies: considerations for genetic counseling

Pre-auricular tags are relatively common isolated congenital anomalies with a prevalence of about 5 per 1000 live births. Several associations with congenital anomalies have been reported and the opportunity of systematic ultrasonography examinations in these patients were debated in the literature. We conducted a retrospective epidemiological study on 95 affected newborns, to evaluate whether infants with pre-auricular tags may be at risk for associated anomalies. Our results focus the attention on the increased risk of congenital urinary tract and heart malformations in newborns with isolated pre-auricular tags. Therefore, we recommend that a carefully genetic clinical examination to evaluated dysmorphic features evocative of a specific pattern or syndrome and an urinary and cardiac ultrasonography should be performed in infants with isolated pre-auricular tags.     

Genomic effects of IFN-beta in multiple sclerosis patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.     

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.     

A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia [corrected

Hereditary spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders for which >/=14 different genetic loci have been identified. In some SCA types, expanded tri- or pentanucleotide repeats have been identified, and the length of these expansions correlates with the age at onset and with the severity of the clinical phenotype. In several other SCA types, no genetic defect has yet been identified. We describe a large, three-generation family with early-onset tremor, dyskinesia, and slowly progressive cerebellar ataxia, not associated with any of the known SCA loci, and a mutation in the fibroblast growth factor 14 (FGF14) gene on chromosome 13q34. Our observations are in accordance with the occurrence of ataxia and paroxysmal dyskinesia in Fgf14-knockout mice. As indicated by protein modeling, the amino acid change from phenylalanine to serine at position 145 is predicted to reduce the stability of the protein. The present FGF14 mutation represents a novel gene defect involved in the neurodegeneration of cerebellum and basal ganglia.     

Gerstmann-Sträussler-Scheinker disease with P102L-V129 mutation: a case with psychiatric manifestations at onset

Gerstmann-Str?ussler-Scheinker disease (GSS) is an adult onset, rare, genetically determined autosomal dominant prion disease. Clinically, it is characterized predominantly by slowly progressive spino-cerebellar dysfunction with ataxia, absent reflexes in the legs and cognitive impairment. Onset is usually in the fifth decade and in the early phase, ataxia is predominant. Mutations in the prion protein gene (PRNP) had been identified and the most important of these is at codon 129. A genotype-phenotype relationship with genetic polymorphism at residue 129 between methionine and valine has been supposed. We describe a patient with GSS and P102L-V129 mutation in which the onset with prominent psychiatric features characterized by apathy and depression and not with cerebellar sign and the clinical course with seizures, nor observed in P102L-V129 cases, allow us to confirm observations that the GSS caused by the 102 mutation is influenced by the codon 129 polymorphism with a specific genotype-phenotype influence, but probably other additional factors might be considered as background for phenotypic variability.     

Genetic variation and population structure in Scandinavian wolverine (Gulo gulo) populations

Wolverine (Gulo gulo) numbers in Scandinavia were significantly reduced during the early part of the century as a result of predator removal programmes and hunting. Protective legislation in both Sweden and Norway in the 1960s and 1970s has now resulted in increased wolverine densities in Scandinavia. We report here the development of 15 polymorphic microsatellite markers in wolverine and their use to examine the population sub-structure and genetic variability in free-ranging Scandinavian wolverine populations as well as in a sample of individuals collected before 1970. Significant subdivision between extant populations was discovered, in particular for the small and isolated population of southern Norway, which represents a recent recolonization. Overall genetic variability was found to be lower than previously reported for other mustelids, with only two to five alleles per locus and observed heterozygosities (H(O)) ranging from 0.269 to 0.376 across the examined populations, being lowest in southern Norway. Analysis of the mitochondrial DNA control region revealed no variation throughout the surveyed populations. As the historical sample did not show higher levels of genetic variability, our results are consistent with a reduction in the genetic variation in Scandinavian wolverines that pre-dates the demographic bottleneck observed during the last century. The observed subdivision between populations calls for management caution when issuing harvest quotas, especially for the geographically isolated south Norwegian population.     

Study of the infectivity of saline-stored Campylobacter jejuni for day-old chicks

The culturability of three Campylobacter jejuni strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.