Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Large-scale preventive chemotherapy for the control of helminth infection in Western Pacific countries: six years later

In 2001, Urbani and Palmer published a review of the epidemiological situation of helminthiases in the countries of the Western Pacific Region of the World Health Organization indicating the control needs in the region. Six years after this inspiring article, large-scale preventive chemotherapy for the control of helminthiasis has scaled up dramatically in the region. This paper analyzes the most recent published and unpublished country information on large-scale preventive chemotherapy and summarizes the progress made since 2000. Almost 39 million treatments were provided in 2006 in the region for the control of helminthiasis: nearly 14 million for the control of lymphatic filariasis, more than 22 million for the control of soil-transmitted helminthiasis, and over 2 million for the control of schistosomiasis. In general, control of these helminthiases is progressing well in the Mekong countries and Pacific Islands. In China, despite harboring the majority of the helminth infections of the region, the control activities have not reached the level of coverage of countries with much more limited financial resources. The control of food-borne trematodes is still limited, but pilot activities have been initiated in China, Lao People's Democratic Republic, and Vietnam.     

The stabilization of locust cuticle

Cuticle from the metathoracic femur of adult locusts (Locusta migratoria) is characterized with respect to changes in water content and in protein extractability during maturation. The swelling behaviour and extractability of fully-sclerotized cuticle are compared to those of chemically-modified, unsclerotized cuticle.It is concluded that although dehydration may contribute to the stabilization of cuticle, it cannot account for the observed differences. The properties of mature cuticle can best be explained by the assumption that covalent cross-links are present between protein molecules.     

Dihydrofurocoumarin glucosides from Angelica archangelica and Angelica silvestris

A water-soluble root extract of Angelica archangelica subsp. litoralis afforded, in addition to adenosine, coniferin and the two known dihydrofurocoumarin glycosides, apterin and 1′-O-β-d-glycopyranosyl-(S)-marmesin (marmesinin), two new dihydrofuranocoumarin glycosides, 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin, and 2′-β-d-glucopyranosyloxymarmesin. For the latter a 2S-configuration was demonstrated, the stereochemistry at position 1′ remaining undefined. Roots of A. silvestris similarly afforded 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin. By correlation with the aglycone 2S,3R)-3-hydroxymarmesin obtained in this work, the absolute configurations (2S,3R) were established for the known dihydrofurocoumarin diesters smirniorin and smirnioridin.     

The mink as an animal model for Pseudomonas aeruginosa adhesion: binding of the bacterial lectins (PA-IL and PA-IIL) to neoglycoproteins and to sections of pancreas and lung tissues from healthy mink

Lung infection with Pseudomonas aeruginosa, leading to chronic lung disease with impaired function, is the major course of morbidity and mortality among cystic fibrosis patients. The bacterium produces two lectins that bind to alpha-D-galactose (PA-IL) and L-fucose (PA-IIL), respectively, and lectin-carbohydrate interactions may be involved in microbial pathogenicity by creating bacterial adherence to epithelial and endothelial cells. An ideal animal model for P. aeruginosa infection has until now not been established, but the mink seems to be the only animal that has been reported to develop spontaneous P. aeruginosa infections in the airways. Since cystic fibrosis also severely may affect pancreatic function, we incubated sections from mink lungs and pancreas with a medium containing Pseudomonas lectins in order to detect in situ binding of the bacterial lectins. In the lungs, both lectins adhered to seromucinous glands located in the submucosa of the larger bronchi. Additionally, PA-IL reacted with the capillaries in the alveolar walls and with the small blood vessels forming the vasa vasorum around the larger vessels, while PA-IIL marked the goblet cells in the bronchial surface epithelium. In the pancreas, both lectins bound to the epithelium in the excretory ducts, and additionally, PA-IL strongly stained the pancreatic capillaries while PA-IIL staining was noticed in the apical part of acinar cells in the exocrine part of the gland while no lectin reaction could be recorded in the endocrine cells. Judging from the results in the present paper the mink should be considered a suitable model to study P. aeruginosa adherence.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

The Homeostatic Chemokine CCL21 Predicts Mortality in Aortic Stenosis Patients and Modulates Left Ventricular Remodeling

Background

CCL21 acting through CCR7, is termed a homeostatic chemokine. Based on its role in concerting immunological responses and its proposed involvement in tissue remodeling, we hypothesized that this chemokine could play a role in myocardial remodeling during left ventricular (LV) pressure overload.

Methods and Results

Our main findings were: (i) Serum levels of CCL21 were markedly raised in patients with symptomatic aortic stenosis (AS, n = 136) as compared with healthy controls (n = 20). (ii) A CCL21 level in the highest tertile was independently associated with all-cause mortality in these patients. (iii) Immunostaining suggested the presence of CCR7 on macrophages, endothelial cells and fibroblasts within calcified human aortic valves. (iv). Mice exposed to LV pressure overload showed enhanced myocardial expression of CCL21 and CCR7 mRNA, and increased CCL21 protein levels. (v) CCR7^−/− mice subjected to three weeks of LV pressure overload had similar heart weights compared to wild type mice, but increased LV dilatation and reduced wall thickness.

Conclusions

Our studies, combining experiments in clinical and experimental LV pressure overload, suggest that CCL21/CCR7 interactions might be involved in the response to pressure overload secondary to AS.