African ancestry is associated with risk of asthma and high total serum IgE in a population from the Caribbean Coast of Colombia

African descended populations exhibit an increased prevalence of asthma and allergies compared to Europeans. One approach to distinguish between environmental and genetic explanations for this difference is to study relationships of asthma risk to individual admixture. We aimed to determine the admixture proportions of a case-control sample from the Caribbean Coast of Colombia currently participating in genetic studies for asthma, and to test for population stratification and association between African ancestry and asthma and total serum IgE levels (tIgE). We genotyped 368 asthmatics and 365 non-asthmatics for 52 autosomal ancestry informative markers, six mtDNA haplogroups and nine haplogroups and five microsatellites in Y chromosome. Autosomal admixture proportions, population stratification, and associations between ancestry and the phenotypes were estimated by ADMIXMAP. The average admixture proportions among asthmatics were 42.8% European, 39.9% African and 17.2% Native American and among non-asthmatics they were 44.2% (P = 0.068), 37.6% (P = 0.007) and 18.1% (P = 0.050), respectively. In the total sample, the paternal contributions were 71% European, 25% African and 4.0% Native American and the maternal lineages were 56.8% Native American, and 20.2% African; 22.9% of the individuals carried other non-Native American mtDNA haplogroups. African ancestry was significantly associated with asthma (OR: 2.97; 95% CI: 1.08–8.08), high tIgE (OR: 1.9; 95% CI: 1.17–3.12) and socioeconomic status (OR = 0.64; 95% CI: 0.47–0.87). Significant population stratification was observed in this sample. Our findings indicate that genetic factors can explain the association between asthma and African ancestry and suggest that this sample is a useful resource for performing admixture mapping for asthma.     

Evaluation of l-glutamine for cryopreservation of boar spermatozoa

The aim of the present study was to evaluate the protective effect of l-glutamine (l-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, l-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk – EY – 20%, and glycerol 3%). No significant improvement (P > 0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility – CASA system – and flow cytometric analysis of plasma and acrosomal membrane integrity −SYBR14/PI/PE-PNA− and plasma membrane stability −M540/YoPro1−). In Experiment 2, l-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P < 0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM l-Gln compared to control (3% glycerol). In Experiment 3, l-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of l-Gln and the absence of EY. In conclusion, l-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.     

Molecular Relationships Between Saccharomyces cerevisiae Strains Involved in Winemaking from Mendoza, Argentina

Three molecular typing techniques were applied to assess the molecular relationships of Saccharomyces cerevisiae strains isolated from winery equipment, grapes, and spontaneous fermentation in a cellar located in “Zona Alta del Río Mendoza” (Argentina). In addition, commercial Saccharomyces strains widely used in this region were also included. Interdelta PCR typing, mtDNA restriction analysis, and microsatellite (SSR) genotyping were applied. Dendrograms were constructed based on similarity among different patterns of bands. The combination of the three techniques discriminated 34 strains among the 35 isolates. The results of this study show the complex relationships found at molecular level among the isolates that share the same ecological environment, i.e., the winemaking process. With a few exceptions, the yeast isolates were generally clustered in different ways, depending on the typing technique employed. Three clusters were conserved independently of the molecular method applied. These groups of yeasts always clustered together and had high degree of similarity. Furthermore, the dendrograms mostly showed clusters combining strains from winery and fermentation simultaneously. Most of the commercial strains included in this study were clustered separately from the other isolates analyzed, and just a few of them grouped with the strains mainly isolated from spontaneous fermentation. Only one commercial strain was clustered repetitively with a noncommercial strain isolated from spontaneous fermentation in the three dendrograms. On the other hand, this study has demonstrated the importance of selecting an appropriate molecular method according to the main objectives of the research.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination

Cytogenetic analysis of 58 boars at an artificial insemination (AI) centre revealed the presence of a reciprocal chromosome translocation, rcp(1;11)(q−;p+), in two Duroc boars. Pedigree analysis of these two boars suggested familial transmission of the chromosome rearrangement. The reproductive consequences of this translocation were determined in a herd of sows that had received semen doses from these and other boars. All sows underwent multiple AI, with different groups established retrospectively depending on the percentage of semen doses provided by the carrier boars ([number of carrier boar doses/total number doses provided] x 100): 0%, 25%, 50%, 75%, 100%. The fertility rates (percentage of successful multiple AIs/total multiple AIs) recorded for multiple AI including semen doses from the carrier boars were not significantly different from those recorded when all semen doses were supplied by normal-karyotype boars. A reduction in litter size of 29.38% was observed, however, in litters sired by one of the carrier boars when its participation in multiple AI was 100%. The number of live-born piglets per litter gradually decreased (P < 0.05) as the percentage participation in multiple AI (25, 50, or 75%) of the carrier boar increased. In addition, both carrier boars sired some piglets with signs of cleft palate and complex malformations of the front legs; these died soon after birth. In conclusion, the boars carrying the translocation rcp(1;11)(q−;p+) showed reduced reproductive performance.     

Hypoglycemic activity of leaf organic extracts from Smallanthus sonchifolius: Constituents of the most active fractions

The aim of the present study was to determine the in vivo hypoglycemic activity of five organic extracts and enhydrin obtained from yacon leaves. The main constituents of the most active fraction were identified. Five organic extracts and pure crystalline enhydrin were administered to normoglycemic, transiently hyperglycemic and streptozotocin (STZ)-diabetic rats. The fasting and post-prandial blood glucose, and serum insulin levels were estimated and an oral glucose tolerance test (OGTT) was performed for the evaluation of hypoglycemic activity and dose optimization of each extract.We found that the methanol, butanol and chloroform extracts showed effective hypoglycemic activity at minimum doses of 50, 10 and 20 mg/kg body weight, respectively, and were selected for further experiments. Oral administration of a single-dose of each extract produced a slight lowering effect in the fasting blood glucose level of normal healthy rats, whereas each extract tempered significantly the hyperglycemic peak after food ingestion. Daily administration of each extract for 8 weeks produced an effective glycemic control in diabetic animals with an increase in the plasma insulin level. Phytochemical analysis of the most active fraction, the butanol extract, showed that caffeic, chlorogenic and three dicaffeoilquinic acids were significant components. Additionally, enhydrin, the major sesquiterpene lactone of yacon leaves, was also effective to reduce post-prandial glucose and useful in the treatment of diabetic animals (minimum dose: 0.8 mg/kg body weight).The results presented here strongly support the notion that the phenolic compounds above as well as enhydrin are important hypoglycemic principles of yacon leaves that could ameliorate the diabetic state.     

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Low expression and instability during isolation are major obstacles preventing adequate structure‐function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C‐terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C‐termini (C_in) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C‐termini (C_out) to C_in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C_out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein‐detergent complex was identified using an extended fluorescence‐detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure‐function studies. Five MPs were successfully cleaved from the GFP tag by site‐specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C_out topology, yielding sufficient protein suitable for structure‐function studies and are superior to expression and purification in the absence GFP fusion tagging.     

High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus

Introduction

Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.

Methods

Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of ³⁵S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, β2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.

Results

Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.

Conclusions

Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.