Extended haplotype analysis of ovine ADRB3 using polymerase chain reaction single strand conformational polymorphism on two regions of the gene

The β3 adrenergic receptor (ADRB3) plays a critical role in the regulation of energy metabolism in mammals. In sheep, intronic polymorphism of the ADRB3 gene has been associated with lamb survival and various production traits. This study investigates variation in the ovine ADRB3 3' untranslated region (3'UTR), a region that may impact expression of the gene. Using PCR- single strand conformational polymorphism (SSCP), six unique patterns (named a-f) were observed in an approximately 304-bp amplicon. Sequencing revealed three single-nucleotide polymorphisms (c.*233A>C, c.*271G>C, c.*357A>T) and a single-nucleotide deletion (c.*257delG). Haplotype analyses showed that the previously described allele A defined by variation in the ovine ADRB3 intron can be divided into three haplotypes (Aa, Ab, and Ac). In total, 16 haplotypes through ovine ADRB3 were detected. This study suggests that ovine ADRB3 is highly polymorphic and that the extended haplotype analysis through the promoter, 5'UTR, coding sequence, intron, and 3'UTR needs to be performed to define the full extent of variation in this gene.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Detection of sequence variation and genotyping of polymorphic genes using polymerase chain reaction stem–loop conformational polymorphism analysis

Here we describe a technique we have named polymerase chain reaction stem–loop conformational polymorphism (PCR–SLCP) analysis for screening DNA sequence variation. This technique uses PCR primers with adapters at the 5′ ends to generate amplicons containing inverted terminal repeat sequences. These enable the formation of specific conformers for single-stranded molecules on denaturation, and sequence variation in the loop region may affect the structure of these and their mobilities during electrophoresis. The technique has the ability to resolve sequence variation in amplicons that cannot be resolved using PCR single-strand conformational polymorphism (PCR–SSCP). PCR–SLCP is simple and sensitive, and the results are highly repeatable.     

Population sinks resulting from degraded habitats of an obligate life-history pathway

In networks of plant–animal mutualisms, different animal groups interact preferentially with different plants, thus forming distinct modules responsible for different parts of the service. However, what we currently know about seed dispersal networks is based only on birds. Therefore, we wished to fill this gap by studying bat–fruit networks and testing how they differ from bird–fruit networks. As dietary overlap of Neotropical bats and birds is low, they should form distinct mutualistic modules within local networks. Furthermore, since frugivory evolved only once among Neotropical bats, but several times independently among Neotropical birds, greater dietary overlap is expected among bats, and thus connectance and nestedness should be higher in bat–fruit networks. If bat–fruit networks have higher nestedness and connectance, they should be more robust to extinctions. We analyzed 1 mixed network of both bats and birds and 20 networks that consisted exclusively of either bats (11) or birds (9). As expected, the structure of the mixed network was both modular (M = 0.45) and nested (NODF = 0.31); one module contained only birds and two only bats. In 20 datasets with only one disperser group, bat–fruit networks (NODF = 0.53 ± 0.09, C = 0.30 ± 0.11) were more nested and had a higher connectance than bird–fruit networks (NODF = 0.42 ± 0.07, C = 0.22 ± 0.09). Unexpectedly, robustness to extinction of animal species was higher in bird–fruit networks (R = 0.60 ± 0.13) than in bat–fruit networks (R = 0.54 ± 0.09), and differences were explained mainly by species richness. These findings suggest that a modular structure also occurs in seed dispersal networks, similar to pollination networks. The higher nestedness and connectance observed in bat–fruit networks compared with bird–fruit networks may be explained by the monophyletic evolution of frugivory in Neotropical bats, among which the diets of specialists seem to have evolved from the pool of fruits consumed by generalists.     

Variation in the ovine hormone-sensitive lipase gene (HSL) and its association with growth and carcass traits in New Zealand Suffolk sheep

The hormone-sensitive lipase (HSL) plays an important role in the regulation of lipolysis in adipose tissues, by catalysing a rate-limiting step in triglyceride hydrolysis. Variation within the human HSL gene (HSL) has been associated with an increased risk of obesity. In this study, variation within three regions (exon 3–4, exon 5–6 and exon 9) of ovine HSL was investigated in 538 Suffolk lambs bred from 13 independent sires using PCR-SSCP. Four sequence variants of intron 5 (designated A–D) and two variants of exon 9 (designated a and b) of ovine HSL were detected. No variation was found in exon 3–4 of the gene. The associations of the variation within ovine HSL with post-weaning growth and carcass traits including eye muscle depth (EMD), eye muscle width (EMW) and fat depth above the eye muscle (FDM) were assessed in 262 of the above 538 lambs using general linear mixed-effects models. In the single variant models, the presence of intron 5 A in a lamb’s genotype was associated with reduced EMD (P = 0.036) and EMW (P = 0.018), whereas the presence of intron 5 C was associated with increased EMD (P < 0.001), EMW (P < 0.001) and FDM (P = 0.017). The association of C with increased EMD (P = 0.002) and EMW (P = 0.002) persisted in the multi-variant model. No association between HSL intron 5 variants and post-weaning growth, or between HSL exon 9 variants, post-weaning growth or carcass traits, were found.