Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.     

铁皮石斛的离体开花

铁皮石斛(Dendrobium candidum),为一种野生兰科植物,在栽培条件下,从种子萌发到开花通常需要3～4a.研究了多种植物激素和多胺对该种石斛组织培养中花芽形成的影响,结果表明在培养基中加入合适浓度的亚精胺(spermidine)或BA(6-苄基腺嘌呤),或同时加入NAA(萘乙酸)和BA均可诱导原球茎或由之形成的无根小苗在3～6个月开花,频率在31.6%～45.8%.当将原球茎在加有ABA(脱落酸)的培养基上预培养后再移到加有BA的培养基上,花芽形成的频率可提高到平均达82.8%(个别实验中可达100%),这种诱导提早开花的现象也与实验材料的发育阶段(原球茎、无根小苗、已生根的小苗)有关,通常发生在根的形成受到完全或部分抑制的情况中.     

ConA激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合

采用DNA-蛋白质体外吸附的方法研究伴刀豆球蛋白激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合.实验结果显示,c-myc与核骨架蛋白的结合具有特异性,在淋巴细胞激活过程中c-myc与P₃₄/P₃₆核骨架蛋白及核纤层蛋白结合,并发生动态变化.     

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.      

PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress

The accumulation of unfolded proteins elicits a cellular response that triggers both pro-survival and pro-apoptotic signaling events. PERK-dependent activation of NF-E2-related factor-2 (Nrf2) is critical for survival signaling during this response; however, the mechanism whereby Nrf2 confers a protective advantage to stressed cells remains to be defined. We now demonstrate that Nrf2 activation contributes to the maintenance of glutathione levels, which in turn functions as a buffer for the accumulation of reactive oxygen species during the unfolded protein response. The deleterious effects of Nrf2 or PERK deficiencies could be attenuated by the restoration of cellular glutathione levels or Nrf2 activity. In addition, the inhibition of reactive oxygen species production attenuated apoptotic induction following endoplasmic reticulum stress. Our data suggest that perturbations in cellular redox status sensitize cells to the harmful effects of endoplasmic reticulum stress, but that other factors are essential for apoptotic commitment.     

Spatial prediction of malaria prevalence in an endemic area of Bangladesh

Background

Malaria is a major public health burden in Southeastern Bangladesh, particularly in the Chittagong Hill Tracts region. Malaria is endemic in 13 districts of Bangladesh and the highest prevalence occurs in Khagrachari (15.47%).

Methods

A risk map was developed and geographic risk factors identified using a Bayesian approach. The Bayesian geostatistical model was developed from previously identified individual and environmental covariates (p < 0.2; age, different forest types, elevation and economic status) for malaria prevalence using WinBUGS 1.4. Spatial correlation was estimated within a Bayesian framework based on a geostatistical model. The infection status (positives and negatives) was modeled using a Bernoulli distribution. Maps of the posterior distributions of predicted prevalence were developed in geographic information system (GIS).

Results

Predicted high prevalence areas were located along the north-eastern areas, and central part of the study area. Low to moderate prevalence areas were predicted in the southwestern, southeastern and central regions. Individual age and nearness to fragmented forest were associated with malaria prevalence after adjusting the spatial auto-correlation.

Conclusion

A Bayesian analytical approach using multiple enabling technologies (geographic information systems, global positioning systems, and remote sensing) provide a strategy to characterize spatial heterogeneity in malaria risk at a fine scale. Even in the most hyper endemic region of Bangladesh there is substantial spatial heterogeneity in risk. Areas that are predicted to be at high risk, based on the environment but that have not been reached by surveys are identified.