Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.     

SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines

Background  

Recent studies of ancestral maize populations indicate that linkage disequilibrium tends to dissipate rapidly, sometimes within 100 bp. We set out to examine the linkage disequilibrium and diversity in maize elite inbred lines, which have been subject to population bottlenecks and intense selection by breeders. Such population events are expected to increase the amount of linkage disequilibrium, but reduce diversity. The results of this study will inform the design of genetic association studies.     

Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

cDNA cloning of translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) from the intertidal harpacticoid copepod Tigriopus japonicus.

We synthesized a cDNA library from the intertidal copepod Tigriopus japonicus, converted it to phagemids and sequenced expressed sequence tags (ESTs). Of these, Tigriopus translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) was further characterized. The Tigriopus TCTP/HRF gene encoded 172 amino acid residues and showed high similarity to Drosophila but moderate similarity to other annelids (e.g. Brugia, Wuchereria and C. elegans). The Tigriopus TCTP/HRF gene appeared in the same clade as the annelids. Here, we describe the analysis of the Tigriopus TCTP/HRF gene.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.