BACKGROUND: It is currently thought that tissue valve degeneration is related to an animal's immune response, which is mainly due to cell surface alpha-Gal epitopes. Cell surface alpha-Gal epitopes are known to be degraded by the enzyme called green coffee bean alpha-galactosidase. It is also well known that alpha-Gal epitopes are immunologically stained by Griffonia Simplicifolia isolectin type B4. We know that many commercially available tissue valves are made of aortic valves and pericardial tissue of pig. So, we investigated whether alpha-Gal epitopes of the aortic valve and pericardial tissue of a pig can be removed by green coffee bean alpha-galactosidase, and we did so by comparing immunologic staining of the tissues before and after the enzyme treatment. MATERIAL AND METHOD: After treating fresh porcine aortic valve and pericardial tissue with green coffee bean alpha-galactosidase at concentrations of 0.5 unit/mL, 1.0 unit/mL, 2.0 unit/mL, respectively, under the condition of pH 6.5, temperature 4degrees C and 24 hours of incubation, each sample was stained with Griffonia Simplicifolia isolectin type B4 immunofluorescent labeling. We then examined whether the alpha-Gal epitopes were reduced or abolished in each consecutive concentration of green coffee bean alpha-galactosidase by comparing the degree of the Griffonia Simplicifolia isolectin B4 staining in each sample. RESULT: In the pig aortic valve tissue, a 1.0 unit/mL concentration of green coffee bean alpha-galactosidase at pH 6.5, 4degrees C and reaction for 24 hours was enough for complete removal of alpha-Gal epitopes from the cell surface on the immunostaining with Griffonia Simplicifolia isolectin B4. On the other hand, more alpha-Gal epitopes were present in the pig pericardial tissue on Griffonia Simplicifolia isolectin B4 staining before the enzyme treatment, and 1.0 unit/mL of galactosidase was not sufficient for complete removal of alpha-Gal from the tissue. 2.0 units/mL of green coffee bean alpha-galactosidase was needed to completely remove the alpha-Gal epitopes from the pericardial tissue on immunostaining. CONCLUSION: The alpha-Gal epitopes of the pig's aortic valve and pericardial tissue were successfully stained with Griffonia Simplicifolia isolectin B4. We could remove nearly all the alpha-Gal epitopes using green coffee bean alpha-galactosidase at the concentration of 1.0 unit/mL in the aortic valve of pig, and 2.0 unit/mL was need to nearly completely remove all the alpha-Gal epitopes in the pericardial tissue of pig under the condition of pH 6.5, 4degrees C and 24 hours of reaction time. In the near future, removal of alpha-Gal epitopes in the pig's aortic valve and pericardial tissue will become a powerful tool for the improvement of the tissue valve durability. It needs to be determined if alpha-galactosidase treated pig tissue is immune to human anti-Gal antibody or anti-Gal monoclonal antibodies.
alpha-Galactosidase ; Aortic Valve ; Coffee ; Epitopes ; Galactosidases ; Griffonia ; Hand ; Humans ; Hydrogen-Ion Concentration ; Lectins ; Pericardium ; Plant Lectins ; Reaction Time ; Tissue Transplantation

AIM: To study the chemical constituents from the seeds of Griffonia simplicifolia Baill. METHODS: The chemical constituents were isolated and purified by a combination of chromatographic materials including silica gel, Sephadex LH-20, and preparative TLC. Their structures were elucidated on the basis of MS and NMR data analysis. The cytotoxic activities were evaluated against HepG2 cancer cell line using the MTT colorimetric method. RESULTS: A new β-carboline alkaloid, griffonine (1), together with seven known alkaloids, hyrtioerectine B (2), 3-carboxy-6-hydroxy-β-carboline (3), hyrtiosulawesine (4), 5-hydroxyindole-3-carbaldehyde (5), 5-hydroxy-3-(2-hydroxyethyl) indole (6), trigonelline (7), and 5-hydroxytryptamine (8) were isolated and identified. Alkaloids 1, 2 and 4 showed growth inhibitory effects on the HepG2 cell line with IC50 values of 23.5, 9.6 and 19.3 μmol·L(-1), respectively. CONCLUSION Alkaloid 1 is new and was named griffonine. Alkaloids 2-7 were isolated from this plant for the first time. Alkaloids 1, 2 and 4 were potentially cytotoxic.
Alkaloids ; chemistry ; isolation & purification ; pharmacology ; Carbolines ; chemistry ; isolation & purification ; pharmacology ; Cell Proliferation ; drug effects ; Griffonia ; chemistry ; Hep G2 Cells ; Humans ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Seeds ; chemistry

In order to test if nestin is a useful marker for various types of progenitor cells, we explored nestin expression in the retina during development. Nestin expression was co-evaluated with bromodeoxyuridine (BrdU) labeling and Griffonia simplicifolia isolectin B4 (GSIB4) histochemistry. Nestin immunoreactivity appears in cell soma of dividing neural progenitor cells and their leading processes in retinas from embryonic day (E) 13 to E20, in accordance with a BrdU-labeled pattern. At postnatal day (P) 5, it is restricted to the end feet of Muller cells. BrdU-labeled nuclei were mainly in the inner part of the inner nuclear layer in postnatal neonates. The retinal vessels demarcated with GSIB4-positive endothelial cells were first distributed in the nerve fiber layer from P3. Afterward the vascular branches sprouted and penetrated deeply into the retina. The endothelial cells positive for GSIB4 and the pericytes in the microvessels were additionally immunoreactive for nestin. Interestingly, the presumed migrating microglial cells showing only GSIB4 reactivity preceded the microvessels throughout the neuroblast layer during vascular sprouting and extension. These findings may suggest that nestin expression represents the proliferation and movement potential of the neural progenitor cells as well as the progenitor cells of the endothelial cell and the pericyte during retinal development. Thus, Muller glial cells might be potential neural progenitor cells of the retina, and the retinal microvasculature established by both the endothelial and the pericyte progenitor cells via vasculogenesis along microglia migrating routes sustains its angiogenic potential.
Bromodeoxyuridine ; Carisoprodol ; Endothelial Cells ; Foot ; Griffonia ; Humans ; Infant, Newborn ; Intermediate Filament Proteins ; Lectins ; Microglia ; Microvessels ; Nerve Fibers ; Nerve Tissue Proteins ; Neurogenesis ; Neuroglia ; Pericytes ; Plant Lectins ; Retina ; Retinal Vessels ; Retinaldehyde ; Stem Cells

Diabetic retinopathy is characterized by the pericyte loss, microaneurysms and neovascularization eventually leads to blindness. The present study was examined changes of the microvasculature histochemically and immunochemically in the diabetic rat retina previously documented neuronal alterations, in order to verify the usefulness of the animal model of diabetes for the pathophysiology of angiogenesis. Diabetic condition was induced by a single intravenous injection of streptozotocin in Sprague-Dawley rats aged of 8weeks. The animals showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, and 12 weeks, respectively. The retinas were processed for Griffonia simplicifolia isolection (GSI) B4 histochmistry, and anti-alpha-smooth muscle actin (alpha-SMA) and anti-NG2 immunochemical techniques. The retinal vasculature was well demarcated by endothelial profiles with GSIB4 histochemistry. alpha-SMA immunoreactivity appeared in the arterioles and the primary capillaries, and NG2 in the arterioles and the whole capillary beds. Changes evoked by diabetes were largely occurred in the capillary. Compared to the retina at normal state, the capillary networks were more complicated, enlarged, and dense. NG2 reactivity was reduced especially under the cytoplasmic processes of the pericytes. In the near periphery of the capillary mainly in the ganglion cell layer of the diabetes, GSIB4 reactive microglia were distributed. These results suggest that the retinal microvasculature showed the precedent events of neovascularization due to diabetes and rat model of diabetes is useful for study of neovascularization mechanism of the diabetic retinopathy.
Actins ; Aged ; Animals ; Arterioles ; Blindness ; Blood Glucose ; Capillaries ; Cytoplasm ; Diabetic Retinopathy ; Ganglion Cysts ; Griffonia ; Humans ; Immunochemistry ; Injections, Intravenous ; Microglia ; Microvessels ; Models, Animal ; Muscles ; Neurons ; Pericytes ; Rats ; Rats, Sprague-Dawley ; Retina ; Retinaldehyde ; Streptozocin