Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo

Synthesis and characterization of CuO nanowires by a simple wet chemical method

We report a successful synthesis of copper oxide nanowires with an average diameter of 90 nm and lengths of several micrometers by using a simple and inexpensive wet chemical method. The CuO nanowires prepared via this method are advantageous for industrial applications which require mass production and low thermal budget technique. It is found that the concentration and the quantity of precursors are the critical factors for obtaining the desired one-dimensional morphology. Field emission scanning electron microscopy images indicate the influence of thioglycerol on the dispersity of the prepared CuO nanowires possibly due to the stabilization effect of the surface caused by the organic molecule thioglycerol. The Fourier transform infrared spectrum analysis, energy dispersive X-ray analysis, X-ray diffraction analysis, and X-ray photoemission spectrum analysis confirm clearly the formation of a pure phase high-quality CuO with monoclinic crystal structure

The Genome of Ganderma lucidum Provide Insights into Triterpense Biosynthesis and Wood Degradation

BACKGROUND: Ganoderma lucidum (Reishi or Ling Zhi) is one of the most famous Traditional Chinese Medicines and has been widely used in the treatment of various human diseases in Asia countries. It is also a fungus with strong wood degradation ability with potential in bioenergy production. However, genes, pathways and mechanisms of these functions are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: The genome of G. lucidum was sequenced and assembled into a 39.9 megabases (Mb) draft genome, which encoded 12,080 protein-coding genes and ∼83% of them were similar to public sequences. We performed comprehensive annotation for G. lucidum genes and made comparisons with genes in other fungi genomes. Genes in the biosynthesis of the main G. lucidum active ingredients, ganoderic acids (GAs), were characterized. Among the GAs synthases, we identified a fusion gene, the N and C terminal of which are homologous to two different enzymes. Moreover, the fusion gene was only found in basidiomycetes. As a white rot fungus with wood degradation ability, abundant carbohydrate-active enzymes and ligninolytic enzymes were identified in the G. lucidum genome and were compared with other fungi. CONCLUSIONS/SIGNIFICANCE: The genome sequence and well annotation of G. lucidum will provide new insights in function analyses including its medicinal mechanism. The characterization of genes in the triterpene biosynthesis and wood degradation will facilitate bio-engineering research in the production of its active ingredients and bioenergy

EMT-Induced Stemness and Tumorigenicity Are Fueled by the EGFR/Ras Pathway

10.1371/journal.pone.0070427PLoS ONE88-POLN

Kaposin-B Enhances the PROX1 mRNA Stability during Lymphatic Reprogramming of Vascular Endothelial Cells by Kaposi's Sarcoma Herpes Virus

Kaposi's sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposi's sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3′-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV

Bladder inflammatory transcriptome in response to tachykinins: Neurokinin 1 receptor-dependent genes and transcription regulatory elements

Background Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). Methods: An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. Results: The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, α-NGF, TSP2). In the absence of NK1R, the matrix Nkx2-5_02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF1, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression. Electrophoretic mobility shift assays confirmed that, in the mouse urinary bladder, activation of NK1R by substance P (SP) induces both NKx-2.5 and NF-kappaB translocations. Conclusion: This is the first report describing a role for Nkx2.5 in the urinary tract. As Nkx2.5 is the unique discriminator of NK1R-modulated inflammation, it can be imagined that in the near future, new based therapies selective for controlling Nkx2.5 activity in the urinary tract may be used in the treatment in a number of bladder disorders

The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4

The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1