We studied the effects of hyperhydricity on subcellular ultrastructure and physiology of leaves during in vitro regeneration of apple plants. Morphological, anatomical and ultrastructural differences between healthy leaf tissues obtained from greenhouse-grown plants and healthy and hyperhydric leaves obtained from shoots raised from nodal shoot explants in a bioreactor were investigated by electron microscopy and confocal laser scanning microscopy. Compared with healthy leaves, hyperhydric leaves showed abnormal, often discontinuous development of the epidermis and cuticle. Stomata were malformed. The leaf lamina appeared thickened and was characterized by poor differentiation between the palisade and spongy mesophyll tissue. Hyperhydric leaves had a significantly lower chloroplast number per cell and chloroplasts showed reduced thylakoid stacking compared with healthy leaves. Hyperhydricity resulted in a general decrease in concentrations of reduced and oxidized pyridine nucleotides, reflecting a reduction in metabolic activity. The activities of antioxidant enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were higher in hyperhydric leaves than in healthy leaves, indicating that hyperhydricity was associated with oxidative stress. Chlorophyll fluorescence measurements provided evidence of oxidative damage to the photosynthetic machinery in hyperhydric leaves: photochemical efficiency of photosystem II, effective quantum efficiency and photochemical quenching were all lower in hyperhydric leaves compared with healthy leaves.
The effects of berberine on long-term administration of L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) were investigated. Rat models of PD were prepared by 6-OHDA lesions in the ipsilateral sides, and then were treated with berberine (5 and 15 mg/kg) and/or L-DOPA (10 mg/kg) once daily for 21 days. Treatments with either concentration of berberine (5 and 15 mg/kg) in 6-OHDA-lesioned groups decreased the numbers of tyrosine hydroxylase (TH)-immunopositive neurons in the substantia nigra and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum as compared to 6-OHDA-lesioned groups. In addition, dopaminergic neuronal cell death of the ipsilateral sides in 6-OHDA-lesioned groups was attenuated by L-DOPA administration. However, both concentrations of berberine in 6-OHDA-lesioned groups treated with L-DOPA aggravated the numbers of TH-immunopositive neurons in the substantia nigra and the levels of dopamine, norepinephrine, DOPAC and HVA in the striatum as compared to rats not treated with berberine. These results suggest that berberine leads to the degeneration of dopaminergic neuronal cells in the substantia nigra in the rat model of PD with chronic L-DOPA administration. Long-term L-DOPA therapy that may involve possibly neurotoxic isoquinoline agents including berberine should involve monitoring for adverse symptoms.
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