Isolation and characterization of phytochelatin synthase in rice seedlings

Rice plants were treated with 50 microM copper sulfate to induce the synthesis of phytochelatins by means of a series of enzymatic reactions, including that of photochelatin synthase. Phytochelatin synthase extracted from 3-week-old rice seedlings was purified through a series of steps including precipitation with ice-chilled acetone, QAE A-50 anion exchange column, Amicon XM-50 ultrafiltration and Polybuffer Exchange (PBE) 94 chromato-focusing. This enzyme had a molecular mass of about 100 kDa with an isoelectric point of 4.0. The temperature and pH optima of this enzyme were 55 degrees C and pH 7.5, respectively. The enzyme was thermal tolerable and unstable under refrigeration at 4 or -20 degrees C. Cadmium was the most effective stimulator, followed by lead, copper, silver, cobalt and other divalent cations. Calcium and magnesium had no effect.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Background

Parkinson’s disease is the second most common neurodegenerative disorders after Alzheimer’s disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration.

Results

Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP⁺) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP⁺ impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP⁺ might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP⁺ caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP⁺ decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP⁺ provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP⁺.

Conclusions

Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson’s disease.     

Amyloid β peptide-mediated neurotoxicity is attenuated by the proliferating microglia more potently than by the quiescent phenotype

Background

The specific role of microglia on Aβ-mediated neurotoxicity is difficult to assign in vivo due to their complicated environment in the brain. Therefore, most of the current microglia-related studies employed the isolated microglia. However, the previous in vitro studies have suggested either beneficial or destructive function in microglia. Therefore, to investigate the phenotypes of the isolated microglia which exert activity of neuroprotective or destructive is required.

Results

The present study investigates the phenotypes of isolated microglia on protecting neuron against Aβ-mediated neurotoxicity. Primary microglia were isolated from the mixed glia culture, and were further cultured to distinct phenotypes, designated as proliferating amoeboid microglia (PAM) and differentiated process-bearing microglia (DPM). Their inflammatory phenotypes, response to amyloid β (Aβ), and the beneficial or destructive effects on neurons were investigated. DPM may induce both direct neurotoxicity without exogenous stimulation and indirect neurotoxicity after Aβ activation. On the other hand, PAM attenuates Aβ-mediated neurotoxicity through Aβ phagocytosis and/or Aβ degradation.

Conclusions

Our results suggest that the proliferating microglia, but not the differentiated microglia, protect neurons against Aβ-mediated neurotoxicity. This discovery may be helpful on the therapeutic investigation of Alzheimer’s disease.     

Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization

Background

Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer''s disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.

Result

Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.

Conclusion

The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer’s disease and atherosclerosis.     

Twist controls skeletal development and dorsoventral patterning by regulating runx2 in zebrafish

Background

Twist1a and twist1b are the principal components of twists that negatively regulate a number of cellular signaling events. Expression of runx2 and downstream targets is essential for skeletal development and ventral organizer formation and specification in early vertebrate embryos, but what controls ventral activity of maternal runx2 and how twists function in zebrafish embryogenesis still remain unclear.

Methodology/Principal Findings

By studying the loss of twist induced by injection of morpholino-oligonucleotide in zebrafish, we found that twist1a and twist1b, but not twist2 or twist3, were required for proper skeletal development and dorsoventral patterning in early embryos. Overexpression of twist1a or twist1b following mRNA injection resulted in deteriorated skeletal development and formation of typical dorsalized embryos, whereas knockdown of twist1a and twist1b led to the formation of abnormal embryos with enhanced skeletal formation and typical ventralized patterning. Overexpression of twist1a or twist1b decreased the expression of runx2b, whereas twist1a and twist1b knockdown increased runx2b expression. We have further demonstrated that phenotypes induced by twist1a and twist1b knockdown were rescued by runx2b knockdown.

Conclusions/Significance

Together, these results suggest that twist1a and twist1b control skeletal development and dorsoventral patterning by regulating runx2b in zebrafish and provide potential targets for the treatment of diseases or syndromes associated with decreased skeletal development.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.