Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.     

Proteomic analysis of the neuroprotective mechanisms of acupuncture treatment in a Parkinson's disease mouse model

Acupuncture is frequently used as an alternative therapy for Parkinson's disease (PD), and it attenuates dopaminergic (DA) neurodegeneration in the substantia nigra (SN) in PD animal models. Using proteomic analysis, we investigated whether acupuncture alters protein expression in the SN to favor attenuation of neuronal degeneration. In C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days, 2 or 100 Hz electroacupuncture (EA) was applied at the effective and specific acupoint, GB34, once a day for 12 consecutive days from the first MPTP treatment. Both treatments in MPTP mice led to restoration of behavioral impairment and rescued tyrosine hydroxylase (TH)-positive DA neurodegeneration. Using peptide fingerprinting MS, we identified changes in 22 proteins in the SN following MPTP treatment, and nine of these proteins were normalized by EA. They were involved in cell death regulation, inflammation, or restoration from damage. The levels of cyclophilin A (CypA), which is a neuroprotective agent, were unchanged by MPTP treatment but were increased in MPTP-EA mice. These results suggest that acupoint GB34-specific EA changes protein expression profiles in the SN in favor of DA neuronal survival in MPTP-treated mice, and that EA treatment may be an effective therapy for PD patients.     

Co-administration of IGF-I and glycosaminoglycans greatly delays motor neurone disease and affects IGF-I expression in the wobbler mouse: a long-term study

The study on wobbler mouse has shown that the combined treatment with low doses of glycosaminoglycans (GAGs) and insulin-like growth factor-I (IGF-I) fully prevented motor neurone death and forelimb impairment up to 9-12 weeks of a mouse's life. The effect was accompanied by the prevention of the early hypertrophy of wobbler neurones, an effect likely due to the promotion of neuronal survival. At the 18th week, wobbler mice treated with IGF-I + GAGs still showed significantly improved forelimb function, reduced muscle atrophy and a higher number of cervical motor neurones. IGF-I alone and GAGs alone were active up to the 3rd week of treatment; thereafter the beneficial effects of single treatments decreased drastically. GAGs and IGF-I treatments also affected IGF-I plasma and muscle levels. In wobbler mice there was a progressive reduction in IGF-I plasma levels that was prevented by IGF-I or GAGs alone and greatly increased, even above heterozygote levels, by the combination treatment. Such a powerful increase was correlated by a small enhancement in insulin-like growth factor binding protein-3 (IGFBP-3) plasma levels, while treatment with IGF-I alone affected very significantly both IGFBP-1 and IGFBP-3. Co-treatment also prevented the decrease in IGF-I content observed in vehicle-treated wobbler mice forelimb muscles.