Evaluating Evolutionary Divergence with Microsatellites

We report the use of microsatellites (MS) to track the recent evolution of swine. Allelic frequencies for nine MS loci linked on swine chromosome 6 (SSC6) representing four western and one Chinese swine breeds were used to estimate genetic distances and times of breed divergence. A phylogenetic tree was constructed which partitioned into western and Meishan breed branches. Yorkshire and Hampshire breeds exhibited the most recent divergence with a calculated distance of 391 years. The oldest divergence, of 2,227 years, was between Meishan and Hampshire swine. Estimates of breed divergence are consistent with historical records. Additional analysis suggests that polymorphic MS linked on a single chromosome are sufficient to determine evolutionary relationships within a single species. Received: 23 September 1996 / Accepted: 26 April 1997     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

Human Disturbance Influences Behaviour and Local Density of Juvenile Frogs

Habitat can be rendered less suitable because of numerous factors, including the presence of humans. Human disturbance is implicated as a threat for hundreds of amphibian species, but there is a dearth of peer‐reviewed literature addressing this topic, and what little is available focuses on adults. Here we present the results of a study examining the effects human disturbance may have on the distribution and behaviour of juvenile Eastern African leaf litter frogs of the genus Arthroleptis. Our findings show that human activity affects the local density of juveniles and strongly influences escape behaviour. These results indicate that costs of human disturbance to juvenile frogs may be severe and that human disturbance may play a role in fragmenting local frog populations.     

Successful elimination of a lethal wildlife infectious disease in nature

Methods to mitigate the impacts of emerging infectious diseases affecting wildlife are urgently needed to combat loss of biodiversity. However, the successful mitigation of wildlife pathogens in situ has rarely occurred. Indeed, most strategies for combating wildlife diseases remain theoretical, despite the wealth of information available for combating infections in livestock and crops. Here, we report the outcome of a 5-year effort to eliminate infection with Batrachochytrium dendrobatidis affecting an island system with a single amphibian host. Our initial efforts to eliminate infection in the larval reservoir using a direct application of an antifungal were successful ex situ but infection returned to previous levels when tadpoles with cleared infections were returned to their natal sites. We subsequently combined antifungal treatment of tadpoles with environmental chemical disinfection. Infection at four of the five pools where infection had previously been recorded was eradicated, and remained so for 2 years post-application.     

Genetic and Informatic Analyses Implicate Kif12 as a Candidate Gene within the Mpkd2 Locus That Modulates Renal Cystic Disease Severity in the Cys1cpk Mouse

We have previously mapped the interval on Chromosome 4 for a major polycystic kidney disease modifier (Mpkd) of the B6(Cg)-Cys1 ^cpk/J mouse model of recessive polycystic kidney disease (PKD). Informatic analyses predicted that this interval contains at least three individual renal cystic disease severity-modulating loci (Mpkd1-3). In the current study, we provide further validation of these predicted effects using a congenic mouse line carrying the entire CAST/EiJ (CAST)-derived Mpkd1-3 interval on the C57BL/6J background. We have also generated a derivative congenic line with a refined CAST-derived Mpkd1-2 interval and demonstrated its dominantly-acting disease-modulating effects (e.g., 4.2-fold increase in total cyst area; p<0.001). The relative strength of these effects allowed the use of recombinants from these crosses to fine map the Mpkd2 effects to a <14 Mbp interval that contains 92 RefSeq sequences. One of them corresponds to the previously described positional Mpkd2 candidate gene, Kif12. Among the positional Mpkd2 candidates, only expression of Kif12 correlates strongly with the expression pattern of Cys1 across multiple anatomical nephron structures and developmental time points. Also, we demonstrate that Kif12 encodes a primary cilium-associated protein. Together, these data provide genetic and informatic validation of the predicted renal cystic disease-modulating effects of Mpkd1-3 loci and implicate Kif12 as the candidate locus for Mpkd2.     

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α₁-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.     

First Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in China: Discovery of Chytridiomycosis in Introduced American Bullfrogs and Native Amphibians in the Yunnan Province,China

Although the chytrid fungus Batrachochytrium dendrobatidis (Bd), the etiological agent of amphibian chytridiomycosis, has been implicated in mass mortality and population declines on several continents around the world, there have been no reports on the presence of Bd infections in amphibians in China. We employed quantitative PCR and histological techniques to investigate the presence of Bd in introduced North American bullfrogs (Rana catesbeiana) (referred to hereafter as bullfrog) and native amphibians in bullfrog-invaded areas of the Yunnan Province, China. A total of 259 samples at five wild sites were collected between June and September in 2007 and 2008, including bullfrogs and four native amphibian species (Rana pleuraden, Rana chaochiaoensis, Odorrana andersonii, and Bombina maxima). In addition, 37 samples of adult bullfrogs were obtained from a food market. Bd infections were discovered in bullfrogs and three native amphibian species from all of the surveyed sites. Of the 39 Bd-positive samples, 35 were from wild-caught bullfrog tadpoles, postmetamorphic bullfrogs, R. pleuraden, R. chaochiaoensis, and O. andersonii, and four were from adult bullfrogs from the market. Our results provide the first evidence of the presence of Bd in Chinese amphibians, suggesting that native amphibian diversity in China is at risk from Bd. There is an urgent need to monitor the distribution of Bd in amphibians in China and understand the susceptibility of native amphibian species to chytridiomycosis. Strict regulations on the transportation of bullfrogs and the breeding of bullfrogs in markets and farms should be drafted in order to stop the spread of Bd by bullfrogs.     

Body proportions of circumpolar peoples as evidenced from skeletal data: Ipiutak and Tigara (Point Hope) versus Kodiak Island Inuit

Given the well‐documented fact that human body proportions covary with climate (presumably due to the action of selection), one would expect that the Ipiutak and Tigara Inuit samples from Point Hope, Alaska, would be characterized by an extremely cold‐adapted body shape. Comparison of the Point Hope Inuit samples to a large (n > 900) sample of European and European‐derived, African and African‐derived, and Native American skeletons (including Koniag Inuit from Kodiak Island, Alaska) confirms that the Point Hope Inuit evince a cold‐adapted body form, but analyses also reveal some unexpected results. For example, one might suspect that the Point Hope samples would show a more cold‐adapted body form than the Koniag, given their more extreme environment, but this is not the case. Additionally, univariate analyses seldom show the Inuit samples to be more cold‐adapted in body shape than Europeans, and multivariate cluster analyses that include a myriad of body shape variables such as femoral head diameter, bi‐iliac breadth, and limb segment lengths fail to effectively separate the Inuit samples from Europeans. In fact, in terms of body shape, the European and the Inuit samples tend to be cold‐adapted and tend to be separated in multivariate space from the more tropically adapted Africans, especially those groups from south of the Sahara. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.