Interchain disulfide bonds in immunoglobulins: analysis by two-dimensional polyacrylamide-gel electrophoresis.

The arrangement of interchain disulfide bonds in immunoglobulins may be determined in a single experiment by two-dimensional SDS-polyacrylamide-gel electrophoresis. The noncovalently associated subunits are first separated on a cylindrical acrylamide-gel by electrophoresis under dissociating conditions. The subunits are then subjected to reduction in situ followed by electrophoresis on a slab gel of acrylamide in a direction perpendicular to the first to separate the constituent polypeptide chains (heavy and/or light chains) of the individual subunits.     

Effects of Calcium and Dairy on Body Composition and Weight Loss in African‐American Adults

Objective: Our objective was to determine the effects of dairy consumption on adiposity and body composition in obese African Americans. Research Methods and Procedures: We performed two randomized trials in obese African‐American adults. In the first (weight maintenance), 34 subjects were maintained on a low calcium (500 mg/d)/low dairy (<1 serving/d) or high dairy (1200 mg Ca/d diet including 3 servings of dairy) diet with no change in energy or macronutrient intake for 24 weeks. In the second trial (weight loss), 29 subjects were similarly randomized to the low or high dairy diets and placed on a caloric restriction regimen (?500 kcal/d). Results: In the first trial, body weight remained stable for both groups throughout the maintenance study. The high dairy diet resulted in decreases in total body fat (2.16 kg, p < 0.01), trunk fat (1.03 kg, p < 0.01), insulin (18.7 pM, p < 0.04), and blood pressure (6.8 mm Hg systolic, p < 0.01; 4.25 mm Hg diastolic, p < 0.01) and an increase in lean mass (1.08 kg, p < 0.04), whereas there were no significant changes in the low dairy group. In the second trial, although both diets produced significant weight and fat loss, weight and fat loss on the high dairy diet were ～2‐fold higher (p < 0.01), and loss of lean body mass was markedly reduced (p < 0.001) compared with the low dairy diet. Discussion: Substitution of calcium‐rich foods in isocaloric diets reduced adiposity and improved metabolic profiles in obese African Americans without energy restriction or weight loss and augmented weight and fat loss secondary to energy restriction.     

Viability of cotton and canola pollen on the proboscis of Helicoverpa armigera: implications for spread of transgenes and pollination ecology

Abstract.  1. Pollen can be transported thousands of kilometres by insects but its viability after long-distance transport is not known. Knowing the potential for this mechanism to cause outcrossing of transgenes from genetically modified (GM) plants is important for risk assessments.
2. The viability of pollen from cotton ( Gossypium hirsutum L.) and canola ( Brassica napus L.) was determined after placing it on the proboscis of Helicoverpa armigera moths for intervals of up to 32 h. Viability of both cotton and canola pollen declined at a much greater rate when in contact with the moth proboscis. Most was non-viable by 8 h compared with 16 h for control cotton pollen or 32 h for canola pollen.
3. There was no significant difference in the rate of decline of pollen viability between the five conventional cotton varieties, or between these and the one GM cotton variety used in these experiments.
4. The number of canola pollen grains remaining on the proboscis declined over time. Very few cotton pollen grains were retained on the proboscis.
5. The reduction in pollen viability during contact with the proboscis might indicate partial ingestion of the pollen via the proboscis.
6. The points above suggest that pollen is unlikely to remain attached or remain viable when carried over large distances by H. armigera . The implications for spread of pollen from transgenic plants and for pollination ecology in general are discussed.     

Multilocus Sequence Typing Supports the Hypothesis that Cow- and Human-Associated Salmonella Isolates Represent Distinct and Overlapping Populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.     

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin

Background

An Atlantic salmon (Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated.

Methods

SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography.

Results

SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands.

Conclusion

SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures.

General Significance

SSL is likely to function as a wide-spectrum pattern recognition protein.