Cloning and Characterization of a Novel Mammalian PP2C Isozyme

PP2C is a structurally diversified protein phosphatase family with a wide range of functions in cellular signal transduction. A novel PP2C subtype, designated PP2Cdelta, was identified from a rat cDNA clone, which encodes a protein of 392 amino acid residues. While PP2Cdelta shares approximately 30% sequence identity in its catalytic domain with the mammalian PP2C, it lacks a 90-residue carboxyl-terminal sequence conserved in mammalian PP2C. Northern blot analysis showed that PP2Cdelta is widely expressed in rat tissues. The transcription of the PP2Cdelta gene was activated in response to stress, such as the addition of ethanol to the culture medium or UV irradiation of cells. Recombinant PP2Cdelta purified from bacteria exhibited a potent Mn2+-dependent serine/threonine phosphatase activity. Unlike other members of the PP2C family, the activity of PP2Cdelta was inhibited, rather than stimulated, by Mg2+. Transfection with PP2Cdelta resulted in inhibition of cell growth, precluding generation of stable 293 or CHO transfectants. Using a modified tetracycline-regulated PP2Cdelta-GFP dicistronic expression cassette, it was revealed that overexpression of PP2Cdelta blocked cell cycle progression and arrested cells at early S phase, resulting in inhibition of DNA synthesis and leading to cell death. These results suggest that PP2Cdelta plays a role in regulation of cell cycle progression via dephosphorylation of its substrates whose appropriate phosphorylation states might be crucial for cell proliferation

SHP-1 Associates with Both Platelet-derived Growth Factor Receptor and the p85 Subunit of Phosphatidylinositol 3-Kinase*

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells

Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli.

A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha.

Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeast Hansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures of H. polymorpha, equivalent to 35-40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however, low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract, it became clear the the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8-9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinant H. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20-22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production

Some factors influencing the proportion of periplasmic hepatitis B virus pre-S2 antigen in the recombinant yeast Hansenula polymorpha.

A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitis B pre-S2 antigen in the recombinant yeast Hansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that it should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast

Protein-tyrosine phosphatase SHP2 is positively linked to proteinase-activated receptor 2-mediated mitogenic pathway.

Proteinase-activated receptor-2 (PAR2), a new member of family of the G protein-coupled receptors, is activated by proteolytic cleavage of its extracellular amino terminus, a mechanism similar to that used by the thrombin receptor. It has been suggested that PAR2 has a potential role in the late phases of the acute inflammatory response and in tissue repair and/or skin-related disorders. Here we demonstrate that the agonist peptide (SLIGRL) stimulated c-fos-mediated mitogenic activation and tyrosine phosphorylation of cellular proteins. One of the tyrosine-phosphorylated proteins was identified as an Src homology-2 domain-containing protein-tyrosine phosphatase, SHP2. The stimulatory effect of the agonist peptide on early gene transcription was markedly blocked by pertussis toxin treatment whereas the induced tyrosine phosphorylation of SHP2 was completely abolished by the drug. More importantly, while expression of wild-type SHP2 enhanced the agonist-stimulatory mitogenic activity, overexpression of a catalytically inactive mutant of SHP2 strongly suppressed the stimulatory effect of the agonist peptide on both early gene transcription and DNA synthesis. These results suggest that SHP2 acts as a positive regulator linked to the PAR2-mediated mitogenic pathway coupled to a pertussis toxin-sensitive heterotrimeric G protein. Demonstration of SHP2 as a positive mediator in a G protein-coupled, receptor-mediated signaling adds to our understanding of the function of both SHP2 and PAR2 in the signaling pathway

Purification and characterization of a protein tyrosine phosphatase containing SH2 domains.

A protein tyrosine phosphatase (PTP) containing two SH2 domains (PTP1C) was purified to near homogeneity from an adenovirus expression system by a two-step chromatographic procedure with a yield of 67%. The purified enzyme behaves as a monomer of 68 kDa on gel filtration and is totally specific for phosphotyrosyl residues. Its optimal pH is around neutrality for protein substrates such as reduced, carboxyamidomethylated, maleylated (RCM)-lysozyme and myelin basic protein but below 5 for low molecular weight compounds such as para-nitrophenyl phosphate (p-NPP) and phosphotyrosine. Furthermore, with the protein substrates, it displays an activity less than 1% of that obtained with other known PTPs but comparable activities toward p-NPP and phosphotyrosine. Its responsiveness toward the usual PTP activators (e.g. spermine) or inhibitors (e.g. vanadate, molybdate, heparin, or Zn2+) varied considerably with the nature of the substrates involved. Limited digestion with trypsin caused the cleavage of a C-terminal segment of the enzyme, giving rise to a 63-kDa fragment; this cleavage resulted in an approximately 20- and 10-fold activation of the enzyme toward RCM-lysozyme and myelin basic protein, respectively

Dilated Dense U-Net for Infant Hippocampus Subfield Segmentation

Accurate and automatic segmentation of infant hippocampal subfields from magnetic resonance (MR) images is an important step for studying memory related infant neurological diseases. However, existing hippocampal subfield segmentation methods were generally designed based on adult subjects, and would compromise performance when applied to infant subjects due to insufficient tissue contrast and fast changing structural patterns of early hippocampal development. In this paper, we propose a new fully convolutional network (FCN) for infant hippocampal subfield segmentation by embedding the dilated dense network in the U-net, namely DUnet. The embedded dilated dense network can generate multi-scale features while keeping high spatial resolution, which is useful in fusing the low-level features in the contracting path with the high-level features in the expanding path. To further improve the performance, we group every pair of convolutional layers with one residual connection in the DUnet, and obtain the Residual DUnet (ResDUnet). Experimental results show that our proposed DUnet and ResDUnet improve the average Dice coefficient by 2.1 and 2.5% for infant hippocampal subfield segmentation, respectively, when compared with the classic 3D U-net. The results also demonstrate that our methods outperform other state-of-the-art methods

Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries

Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targeting the MVP gene (siMVP) and one siRNA targeting the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs