Insulin‐Like Growth Factor‐1 Regulates the Expression of Luteinizing Hormone Receptor and Steroid Production in Bovine Granulosa Cells

Luteinizing hormone LH plays important roles in follicular maturation and ovulation. The effects of LH are mediated by LH receptor (LHR) in the ovary. However, the factors that regulate the expression of LHR in bovine granulosa cells (GCs) are not well known. Insulin‐like growth factor‐1 (IGF‐1) is known to play a key role in the acquisition and maintenance of functional dominance. To better understand the roles of LHR expression and IGF‐1, we conducted three experiments to determine (i) mRNA expression of LHR in the GCs of developing follicles, (ii) the effects of IGF‐1 on LHR mRNA expression in cultured GCs and (iii) the effects of IGF‐1 on estradiol (E2), progesterone (P4) and androstenedione (A4) production by non‐luteinized GCs. In experiment 1, small follicles (<6 mm Ø) expressed lower levels of LHR than mid‐sized follicles (6–8 mm Ø) and large follicles (≥9 mm Ø) expressed the highest levels of LHR mRNA (p < 0.05). In experiment 2, IGF‐1 (1 and 100 ng/ml) increased (p < 0.05) the expression of LHR mRNA in GCs from small and large follicles. In experiment 3, IGF‐1 (0.1–100 ng/ml) increased A4 and E2 in GCs from both small and large follicles but increased P4 only in large follicles. IGF‐1 in combination with LH (0.1 and 1 ng/ml) increased P4 and A4 in large follicles, and increased E2 and A4 in GCs of small follicles. These findings strongly support the concept that IGF‐1 upregulates LHR mRNA expression as well as A4 and E2 production in GCs and that IGF‐1 is required for determining which follicle becomes dominant and acquires ovulatory capacity.     

Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens

World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus and chicken CD154 (HACD) is tested with the aim of enhancing humoral and cellular immune responses in chickens. Protein expression was carried out by transducing several mammalian cell lines with recombinant adenoviral vectors. HACD purification was assessed by three distinct purification protocols: immunoaffinity chromatography by elution at acidic pH or with a chaotropic agent and size exclusion chromatography. Humoral and cellular immune responses were measured using the hemagglutination inhibition assay and the semiquantitative real time PCR, respectively. The results showed that humoral response against HACD was significantly higher than the obtained with HA alone after booster (P<0.01, P<0.05). From HACD molecules purified by distinct protocols, only the obtained by size exclusion chromatography generated hemagglutinationin-inhibition activity. IFN-γ levels indicated that cellular immune response was significantly higher with HACD, in its pure or impure form, compared to its counterpart HA (P<0.01). These data demonstrate that HACD is able to significantly enhance humoral and cellular immune responses against HA antigen, which make this fusion protein a promising subunit vaccine candidate against H5N1 virus outbreaks.     

Genetic relationships among local potato cultivars from Spain using SSR markers

Microsatellite markers (SSR) were used to fingerprint a total of 105 local potato cultivars from Spain. A set of 41 cultivars from Tenerife Island, 19 from the island of La Palma, and 45 local varieties from peninsular Spain were analysed. Some of these varieties represent relicts of the early introductions originating from South America and have been characterised previously morphologically and ecophysiologically. We observed within our materials a total of 76 SSR alleles. Only seven of them were present in all varieties. Several accession and group specific alleles were detected. Similarity coefficients were computed from the molecular data and cluster analyses were performed. The obtained dendrogram was generally in good agreement with previous classifications of the accessions as Solanum tuberosum L. subsp. andigena (Juz. et Bukasov) Hawkes S. tuberosum L. subsp. tuberosum and S. chaucha Juz. et Bukasov genotypes. Also cultivar groups with identical or related common names showed the same SSR patterns or clustered closely together. In addition we performed Principal Coordinate Analysis with the set of genotypes. Results of both analysis methods were generally in good agreement, but also some smaller differences were detected in the associations of groups and genotypes. According to the molecular patterns for some accessions misleading or confounded names were evident, and in some cases the molecular patterns showed also discrepancies with previous species assignments, suggesting the need for a more detailed comparative study of these accessions.     

Cytokinin-dependent improvement in transgenic P(SARK)::IPT tobacco under nitrogen deficiency

Wild-type (WT) and transgenic tobacco plants overexpressing isopentenyltransferase (IPT), a gene coding the rate-limiting step in cytokinin (CKs) synthesis, were grown under limited nitrogen (N) conditions to evaluate the role of CKs in NUE (N-use efficiency) and in different parameters that determine the quality of tobacco leaves. The results indicate that WT tobacco plants submitted to N deficiency show a decline in the leaf/root ratio, associated with a decrease in the NUE and in tobacco-leaf quality, defined by an increase in the quantity of nicotine. On the contrary, the transgenic plants submitted to N deficiency maintained the leaf/root ratio, presenting a higher NUE and greater quality of tobacco leaves than the WT plants, as the latter showed reduced nicotine and an increase in reducing sugars under severe N-deficiency conditions. Therefore, the overexpression of CKs under N deficiency could be a useful tool to improve tobacco cultivation, given that it could reduce N-fertilizer application and thereby provide economic savings and environmental benefits, maintaining yield and improving tobacco leaf quality.     

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.

Results

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.

Conclusions

Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M₃ seed of Brassica rapa from the RevGenUK TILLING service.     

Detection of Enterohemorrhagic Escherichia coli in Meat Foods using DNA Probes,Enzyme‐Linked Immunosorbent Assay and Polymerase Chain Reaction

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhoea with blood and haemolytic uremic syndrome (HUS) in children and elderly people. Infections with EHEC are a world‐wide public health problem, related to consumption of contaminated ground beef. The aim of this study was to establish whether different meat foods sold in Santiago, Chile pose an infection risk by EHEC and to evaluate three different diagnostic techniques in foods, to determine which is most applicable for use in Chile. A parallel analysis was performed on 64 samples of meat foods (23 refrigerated ground meat, 23 refrigerated long pork sausages and 18 frozen hamburgers) sold in Santiago, Chile using DNA probes, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Twenty‐four samples (24 of 64=37.5 %) were positive by DNA probes, ELISA or PCR. The positive and negative predictive values, sensitivity and specificity of ELISA were 26.7, 81.6, 30.8 and 78.4 %, respectively. The positive and negative predictive values, sensitivity and specificity of PCR were 91.7, 96.2, 84.6 and 98 %, respectively. The EHEC serogroups most frequently isolated were O158, O157, O119, O125 and O114. These results show that, although molecular techniques such as enzyme immunoassays are useful for EHEC detection in meat foods, PCR has advantages in terms of sensitivity, specificity, cost and ease of implementation in Chile.     

Selection for postweaning gain in rats: III. Correlated response in maternal performance

Effects from 34 generations of selection, either up (U) or down (D), for 3- to 9-wk weight gain on genetic direct and postnatal maternal effects, dam size and maternal efficiency were evaluated in 25 cross-fostered sets of rats. Direct genetic effects on 12-d pup weight were 14 and 9% above controls (C) for U and D lines, respectively (P less than .01). Postnatal maternal effects on 12-d pup weight were also 11% higher for the U line (P less than .01), but not different from C for the D line. Females of the U line were heavier (P less than .01) by 13% at mating, 12% at parturition and 15% at 12 d of lactation, relative to controls. They also produced 11% greater total litter weight at 12 d and consumed 9% more feed, but were only nonsignificantly lower in feed per unit of pup 12-d litter weight. Females of D line were -8, -7 and -3% relative to controls in the three weights, but did not differ from controls in 12-d litter weight, feed consumed or in feed efficiency during lactation.