ABSTRACT
Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.
Subject(s)
Bauhinia/chemistry , Chloroplasts/chemistry , Diabetes Mellitus, Experimental/drug therapy , Hypoglycemic Agents/isolation & purification , Insulin-Like Growth Factor Binding Proteins/isolation & purification , Plant Leaves/chemistry , Animals , Autoantibodies/blood , Bauhinia/cytology , Cattle , Chloroplasts/ultrastructure , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Hypoglycemic Agents/therapeutic use , Immunoglobulin G/blood , Insulin-Like Growth Factor Binding Proteins/therapeutic use , Mice , Microscopy, Electron, Transmission , Plant Leaves/cytologyABSTRACT
One the most important aspects of a biosensor is related to immobilization and maintenance of specific reference compounds on sensing surfaces. A method for the immobilization of polysaccharides to a silicon oxide surface intended for Surface Acoustical Waves (SAW) sensors is described. Silicon oxide is a hydrophobic inorganic support used for the fabrication of many electronic devices. The pneumococcal polysaccharide (PPS) vaccine is immobilized via Protein A after pre-treatment of the surface with hydrochloric acid. The effects of non-specific binding are discussed. The results indicate that the immobilization of PPS via Protein A increases the sensitivity of detecting Streptococcus pneumoniae antibodies in human sera and offers greater reproducibility of response compared with ELISA methods. The principles of this technique are simple and are applicable to the immobilization of many capsular polysaccharides.
Subject(s)
Biosensing Techniques/methods , Acoustics , Antibodies, Bacterial/blood , Biosensing Techniques/statistics & numerical data , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Humans , Pneumococcal Vaccines , Polysaccharides, Bacterial , Sensitivity and Specificity , Silicon Dioxide , Streptococcus pneumoniae/immunologyABSTRACT
We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.
Subject(s)
Fungi/chemistry , Insulin/analysis , Plant Proteins/analysis , Proteins/analysis , Rhodophyta/chemistry , Animals , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Blotting, Western , Cattle , Cyanobacteria/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fungi/genetics , Molecular Weight , Plant Proteins/genetics , Rhodophyta/geneticsABSTRACT
Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15 percent SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.
Subject(s)
Animals , Cattle , Mice , Bauhinia/chemistry , Chloroplasts/chemistry , Diabetes Mellitus, Experimental/drug therapy , Hypoglycemic Agents/isolation & purification , Insulin-Like Growth Factor Binding Proteins/isolation & purification , Plant Leaves/chemistry , Autoantibodies/blood , Bauhinia/cytology , Chromatography, High Pressure Liquid , Chloroplasts/ultrastructure , Electrophoresis, Polyacrylamide Gel , Hypoglycemic Agents/therapeutic use , Immunoglobulin G/blood , Insulin-Like Growth Factor Binding Proteins/therapeutic use , Microscopy, Electron, Transmission , Plant Leaves/cytologyABSTRACT
We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution