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Oyster mushrooms (genus Pleurotus ) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae . Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse (61.5 and 46.2%, respectively). In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Ji, Cheng, Bau, Dong and Gao.)

The amino acid sequence enriched with proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) is a signal-transducing agent providing unique features to its substrate nuclear proteins (PEST-NPs). The PEST motif is responsible for particular posttranslational modifications (PTMs). These PTMs impart distinct properties to PEST-NPs that are responsible for their activation/inhibition, intracellular localization, and stability/degradation. PEST-NPs participate in cancer metabolism, immunity, and protein transcription as oncogenes or as tumor suppressors. Gene-based therapeutics are getting the attention of researchers because of their cell specificity. PEST-NPs are good targets to explore as cancer therapeutics. Insights into PTMs of PEST-NPs demonstrate that these proteins not only interact with each other but also recruit other proteins to/from their active site to promote/inhibit tumors. Thus, the role of PEST-NPs in cancer biology is multivariate. It is hard to obtain therapeutic objectives with single gene therapy. An especially designed combination gene therapy might be a promising strategy in cancer treatment. This review highlights the multifaceted behavior of PEST-NPs in cancer biology. We have summarized a number of studies to address the influence of structure and PEST-mediated PTMs on activation, localization, stability, and protein-protein interactions of PEST-NPs. We also recommend researchers to adopt a pragmatic approach in gene-based cancer therapy., (© 2020 Wiley Periodicals LLC.)

Lung cancer, especially lung adenocarcinoma, is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes. Consistently, it was directly associated with the poor prognosis of lung adenocarcinoma patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion, and migration in vitro , and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo . Mechanically, DSTN associates with and facilitates nuclear translocation of β-catenin, which promotes epithelial-to-mesenchymal transition (EMT). Taken together, our results indicated that DSTN enhances lung cancer malignancy through facilitating β-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of lung adenocarcinoma. IMPLICATIONS: This finding indicates that DSTN facilitates β-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma., (©2020 American Association for Cancer Research.)

Background: pH-sensitive peptides are a relatively new strategy for conquering the poor endosomal release of cationic polymer-mediated transfection. Modification of antimicrobial peptides by exchanging positively-charged residues with negatively-charged glutamic acid residues (Glu) greatly improves its lytic activity at the endosomal pH, which could improve cationic polymer-mediated transfection., Methods: In the present study, we investigated the effect of the number of Glu substituted for positively-charged residues on the endosomal escape activity of AR-23 and the ability of mutated AR-23 with respect to enhancing cationic polymer-mediated transfection. Three analogs were synthesized by replacing the positively-charged residues in the AR-23 sequence with Glu one-by-one., Results: The pH-sensitive lysis ability of the peptides, the effect of peptides on the physicochemical characteristics, the intracellular trafficking, the transfection efficiency and the cytotoxicity of the polyplexes were determined. Increased lytic activity of peptides was observed with the increased number of Glu replacement in the AR-23 sequence at acidic pH. The number of Glu substituted for positively-charged residues of AR-23 dramatically affects its lysis ability at neutral pH. Triple-Glu substitution in the AR-23 sequence greatly improved poly(l-lysine)-mediated gene transfection efficiency at the same time as maintaining low cytotoxicity., Conclusions: The results indicate that replacement of positively-charged residues with sufficient Glu residues may be considered as a method for designing pH-sensitive peptides, which could be applied as potential enhancers for improving cationic polymer-mediated transfection., (© 2020 The Authors. The Journal of Gene Medicine published by John Wiley & Sons Ltd.)

Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan sulfate and hence participates in remodeling of the extracellular matrix, leading to release of cytokines that are immobilized by binding to heparan sulfate proteoglycans (HSPGs), and consequently activating signaling pathways. This function of HPSE is correlated to its expression level that is normally very low in majority of the tissues. Exceptionally, human platelets express high level of HPSE, suggesting a unique physiological role in this cell. Using K562 cell line, we found a progressive increase of HPSE during the megakaryocytic differentiation. Analysis of a series of megakaryocytic differentiation-related heparin-binding proteins (HBPs) in the cell culture medium revealed an exclusive positive correlation between the level of interleukin 6 (IL-6) and HPSE expression. IL-6 modulated megakaryocytic differentiation through activation of STAT3. Further, we demonstrated that overexpression of HPSE potentiates megakaryocytic differentiation, whereas elimination of HPSE led to a delayed differentiation. This function of HPSE is associated with its activity, as overexpression of inactive HPSE had no effect on IL-6 production and megakaryocytic differentiation. The role of HPSE is further supported by the observation in an umbilical cord blood CD34+ cells megakaryocytic differentiation model. Our data propose a novel role for HPSE in platelets production by a HPSE/IL-6/STAT3 positive feedback loop that specifically regulates megakaryocytes maturation., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Objective: This study aims to investigate whether PM2.5 exposure is involved in the induction of alveolar epithelial cell apoptosis and the progression of emphysema in mice, and to further explore its specific molecular mechanism., Materials and Methods: A certain number of PM2.5 exposed mice and normal control mice were selected, and a lung resection operation was performed to collect the pulmonary tissue samples, which were then analyzed by hematoxylin and eosin (H&E) staining assay. Subsequently, the total protein in the pulmonary tissues of mice in PM2.5 exposure group and control group was extracted, and the p53 protein level was detected by Western blot. Meanwhile, in A549 cells, after treatment of different doses of PM2.5, the protein levels of p53, caspase3, and clv-caspase3 were examined by Western blot while the mRNA levels of p53, Siva-1, and clv-caspase3 were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. In addition, flow cytometry was carried out to measure the incidence of cell apoptosis, while chromatin immunoprecipitation (ChIP) assay was performed to verify whether p53 binds to the Siva-1 promoter region and thus regulates its transcription process., Results: H&E staining revealed that PM2.5 exposure caused pathological damage in the pulmonary tissues and the expansion of the spatial structure of alveoli, which led to emphysema in mice. Moreover, p53 protein expression in pulmonary tissue of mice in PM2.5 exposure group was remarkably higher than that in the control group. Subsequently, A549 cells were treated with 0, 25, 50, 100 μg/ml PM2.5 for 48 h, and it was found that, with the increase of PM2.5 exposure dose, the p53 protein level, Siva-1 mRNA level and cell apoptosis rate were all found increased in a dose-dependent manner, which could be partially reversed by transfection of si-p53 in A549 cells. In addition, CHIP experiments confirmed that p53 can bind to the Siva-1 promoter region and directly regulate Siva-1 transcription. In A549 cells, PM2.5 exposure increased the expression of the clv-caspase3 protein, which was reversed by the knockdown of p53; however, simultaneous overexpression of Siva-1 could further increase the clv-caspase3 protein level. Additionally, flow cytometry also revealed that PM2.5 exposure induced apoptosis of alveolar epithelial cells, while the knockdown of p53 reduced that, which could be promoted by the overexpression of Siva-1., Conclusions: PM2.5 exposure can promote the transcription of Siva-1 to induce apoptosis of alveolar epithelial cells and accelerate the progression of emphysema in mice by enhancing p53 protein expression.

Polypeptide sequences enriched with proline (P), glutamic acid (E), aspartic acid (D) and serine (S)/ threonine (T) (PEST) have been reported to be the most abundant and frequently distributed at the cellular level. There is growing evidence that PEST sequences act as proteolytic recognition signals for degradation of residual proteins which is critical for activation or deactivation of regulatory proteins involved in cellular signaling pathways of cell growth, differentiation, stress responses and physiological death. A PEST containing nuclear protein (PCNP) was demonstrated as a tumor suppressor in a neuroblastoma cancer model and tumor promoter in lung adenocarcinoma cancer model. Its unique properties like ubiquitination by NIRF, co-localization with NIRF in nucleus and tumor progression attract the attention of researchers. PCNP was reported to be ubiquitinated by ring finger protein NIRF in E3 ligase manner and as modulator of MAPK and PI3K/AKT/mTOR signaling pathways. In this review, we summarize PCNP linked DNA damage response, Post translational modifications, and transportation to address initiation, prognosis, and resistance of tumor cells in terms of cell cycle regulation, transcription and apoptosis. Hence, we demonstrate PCNP as a novel target in cancer diagnosis and treatment., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objective: To explore the anti-myeloma effect of suberoylanilide hydroxamic acid (SAHA) and on mouse myeloma cell line SP2/0 in vitro and in vivo and its mechanism., Methods: The inhibitory effect of SAHA on SP2/0 cells was measured by CCK-8 assay,and the apoptosis and cell cycle were analyzed by flow cytometry FACS. The protein expression of Caspase-3 and p53 of SP2/0 cells treated with SAHA were examined by Western blot. Annexin V/7-AAD double staining was performed to detect the apoptosis of SP2/0 induced by SAHA in vitro. SP2/0 cells (1×10 6 ) resuspended in 200 µl PBS were inoculated subcutaneously and intravenously into BALB/c mice, so as to establish aggressive or non-aggressive myeloma-bearing mouse models respectively. On day 3 after modeling, mice received SAHA or vehicle control treatment by intraperitoneal injection. The dose of SAHA was 60 mg/kg·d, 5 times a week for 3 weeks., Results: In SAHA-treated SP2/0 cells, the proliferation inhibition rate and apoptotic cells increased in a dose dependent manner. Also, SAHA significantly increased the ratio of cells in G 2 phase and decreased in S phase. Molecular mechanisms of apoptosis and cell cycle arrest of SP2/0 induced by SAHA partly correlated with up-regulating the expression level of Caspase-3 and p53. In the non-aggressive myeloma-bearing mice, SP2/0 cells disappeared in peripheral blood after SAHA treatment. In the aggressive myeloma-bearing mice, inhibition of tumor growth and prolongation of the cell survival were observed after SAHA treatment., Conclusion: SAHA inhibited SP2/0 cell proliferation, this effect associates with inducing apoptosis and cell cycle arrest, the mechanism of SAHA ralates partly with activating Caspase-3 and p53 pathway.

Background: The tumor acidic microenvironment, a common biochemical event in solid tumors, offers evolutional advantage for tumors cells and even enhances their aggressive phenotype. However, little is known about the molecular mechanism underlying the acidic microenvironment-induced invasion and metastasis., Methods: We examined the expression of the acid-sending ion channel (ASIC) family members after acidic exposure using RT-PCR and immunofluoresence. Gene manipulation was applied to reveal the potential of ASIC2 on invasion, proliferation, colony formation of colorectal cancer (CRC). We assessed the in vivo tumor growth by subcutaneous transplantation and metastasis by spleen xenografts. Chromatin immunoprecipitation-sequencing was used to uncover the binding sites of NFAT1. Finally, we examined the expression of ASIC2 in CRC tissues using immunohistochemistry., Results: Acidic exposure led to up-regulation of the acid-sensing ion channel, ASIC2, in colorectal cancer (CRC) cells. ASIC2 overexpression in CRC cell lines, SW480 and HCT116, significantly enhanced cell proliferation in vitro and in vivo, while ASIC2 knockdown had the reverse effect. Importantly, ASIC2 promoted CRC cell invasion under acidosis in vitro and liver metastasis in vivo. Mechanistically, ASIC2 activated the calcineurin/NFAT1 signaling pathway under acidosis. Inhibition of the calcineurin/NFAT pathway by cyclosporine A (CsA) profoundly attenuated ASIC2-induced invasion under acidosis. ChIP-seq assay revealed that the nuclear factor, NFAT1, binds to genes clustered in pathways involved in Rho GTPase signaling and calcium signaling. Furthermore, immunohistochemistry showed that ASIC2 expression is increased in CRC samples compared to that in adjacent tissues, and ASIC2 expression correlates with T-stage, distant metastasis, recurrence, and poor prognosis., Conclusion: ASIC2 promotes metastasis of CRC cells by activating the calcineurin/NFAT1 pathway under acidosis and high expression of ASIC2 predicts poor outcomes of patients with CRC.

Chiral diamine catalysts 11a-e derived from α,α-diphenyl prolinol were prepared and successfully applied to the Michael addition of aromatic oximes to α,β-unsaturated aldehydes in mediocre to good yields (up to 78%) and good to high enantioselectivities (up to 93% ee)., (© 2017 Wiley Periodicals, Inc.)

Background: Poor endosomal release is a major barrier of polyplex-mediated gene transfection. Antimicrobial peptides (AMPs) are commonly used to improve polyethylenimine (PEI)-mediated gene transfection by increasing endosomal release. In the present study, we designed novel pH-sensitive peptides that highly enhance transfection efficiency compared to their parent peptides., Methods: Two analogues of melittin (Mel) and RV-23 (RV) were synthesized by replacing the positively-charged residues in their sequences with glutamic acid residues. The pH-sensitive lysis ability of the peptides, the effect of the peptides on physicochemical characteristics, the intracellular trafficking, the transfection efficiency, and the cytotoxicity of the polyplexes were determined., Results: The acidic peptides showed pH-sensitive lytic activity. The hemolytic activity of acidic peptides at pH 5.0 was higher than that at pH 7.4. The incorporation of acidic peptides did not affect the DNA binding ability of PEI but affected the physicochemical characteristics of the PEI/DNA polyplexes, which may be beneficial for endosomal release and gene transfection. The incorporation of acidic peptides into PEI/DNA polyplexes enhanced the PEI-mediated transfection efficiency corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone., Conclusions: The results of the present study indicate that replacement of positively-charged residues with glutamic acid residues in the AMP sequence yields pH-sensitive peptides, which enhance the transfection efficiency of PEI/DNA polyplexes in various cell lines., (Copyright © 2017 John Wiley & Sons, Ltd.)

Objective: To investigate the efficacy and safety of the recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein (rhTNFR: Fc, etanercept) for the treatment of occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) . Methods: In September 2011 to February 2016, 12 patients with OMLDT were treated with etanercept 25 mg, subcutaneous injection, twice per week, doubling of first dose. The course of treatment was 6 weeks. The drug eruption area and severity index (DASI) score, the proportion of patients achieving a 50%, 75% and 90% reduction in DASI (DASI50, DASI75, DASI90) and the serum level of TNF-α were used to assess the efficacy at different times. Adverse reactions were also recorded and evaluated. The results were statistically analyzed by nonparametric Friedman test and repetitive measurement ANOVA using the software SPSS19.0. Results: After 4 weeks treatment, the DASI score decreased form 56.33±7.02 to 0.50±0.91 ( P <0.01) . The DASI50, DASI75 and DASI90 were all increased to 12 (100%) . The serum level of TNF-α decreased form (43.74±41.62) pg/ml to (3.03±0.47) pg/ml ( P <0.01) . Statistically significant difference was observed from the above indexes. There were no adverse reactions in clinical application. Conclusion: Recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein may be a safe and effective drug in the treatment of OMLDT.

Pancreatic cancer (PC), characterized by aggressive local invasion and metastasis, is one of the most malignant cancers. Gemcitabine is currently used as the standard drug for the treatment of advanced and metastatic PC, but with limited efficacy. In this study, we demonstrated that gemcitabine increased the expression of heparanase (HPA1), the only known mammalian endoglycosidase capable of cleaving heparan sulfate, both in vitro and in vivo . Furthermore, overexpression of HPA1 in PC cell lines enhanced proliferation and invasion, accompanied with elevated phosphorylation of EGFR. In addition, we showed that the NF-κB pathway mediated the gemcitabine-induced HPA1 expression. Importantly, we found that an HPA1 inhibitor attenuated gemcitabine-induced invasion of PC cells. Finally, we showed that HPA1 was of negative prognostic value for PC patients. Taken together, our results demonstrated that gemcitabine-induced HPA1 promotes proliferation and invasion of PC cells through activating EGFR, implying that HPA1 may serve as promising therapeutic target in the treatment of PC., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

RV-23 is a melittin-related antibacterial peptide (MRP) with lower cytotoxicity than either melittin or AR-23, another MRP. The aim of this study was to explore the mechanism of RV- 23's antibacterial selectivity and its hemocompatibility. The results showed that all the peptides exhibited lytic activity against Staphylococcus aureus and Escherichia coli, with RV-23 showing the highest potency. Moreover, RV-23 had lower cytotoxicity than melittin or AR-23 at their minimal inhibitory concentration. In addition, CD experiments showed that melittin, RV-23, and AR-23 all had a typical α-helical structure, and RV-23 had the lowest α-helix content. The structural information showed that RV-23 has the lowest hydrophobicity and highest hydrophobic moment. Because hydrophobicity and α-helix content are believed to correlate with hemolysis, the results indicate that the selective lytic activity against bacteria of RV-23 may be due to its low hydrophobicity and α-helicity, which lead to low cytotoxicity without affecting antibacterial activity. Furthermore, RV-23 did not affect the structure and function of blood components such as red blood cells, platelets, albumin, and the blood coagulation system. In conclusion, RV-23 is a cell-selective antibacterial peptide with high hemocompatibility due to its unique structure.

AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents.

Background: Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with α-galactosidase and α-N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro., Materials and Methods: A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na(+), and free K(+). The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual α-galactosidase or α-N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility., Results: The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either α-galactosidase or α-N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test., Discussion: The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion.

The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.

Background: The increase in mobile phone use has generated concerns about possible risks to human health, especially the development of brain tumors. Whether tumor patients should continue to use mobile telephones has remained unclear because of a paucity of information. Herein, we investigated whether electromagnetic fields from mobile phones could alter the biological features of human tumor cells and act as a tumor-promoting agent., Methods: Human glioblastoma cell lines, U251-MG and U87-MG, were exposed to 1950-MHz time division-synchronous code division multiple access (TD-SCDMA) at a specific absorption rate (maximum SAR = 5.0 W/kg) for 12, 24, and 48 h. Cell morphologies and ultra-structures were observed by microscopy and the rates of apoptosis and cell cycle progression were monitored by flow cytometry. Additionally, cell growth was determined using the CKK-8 assay, and the expression levels of tumor and apoptosis-related genes and proteins were analyzed by real-time PCR and western blotting, respectively. Tumor formation and invasiveness were measured using a tumorigenicity assay in vivo and migration assays in vitro., Results: No significant differences in either biological features or tumor formation ability were observed between unexposed and exposed glioblastoma cells. Our data showed that exposure to 1950-MHz TD-SCDMA electromagnetic fields for up to 48 h did not act as a cytotoxic or tumor-promoting agent to affect the proliferation or gene expression profile of glioblastoma cells., Conclusions: Our findings implied that exposing brain tumor cells in vitro for up to 48 h to 1950-MHz continuous TD-SCDMA electromagnetic fields did not elicit a general cell stress response.

Suppressor of cytokine signaling 3 (SOCS3) plays an important role in mice fetal liver erythropoiesis, but the roles of SOCS3 in human hematopoietic stem cells (HSCs) have not been well investigated. In the present study, lentiviral small interference RNA expression vectors (shRNA) of SOCS3 were constructed and stably transferred into HSCs. We found that SOCS3 knockdown induced erythroid expansion in HSCs. Conversely, Ectopic expression of SOCS3 in progenitor cells blocked erythroid expansion and erythroid colony formation of HSCs. To further explore the involved mechanism, we compared gene expression profiles of SOCS3-shRNA tranduced HSCs with that of control HSCs by whole genome microarrays. The results indicated that cell developmental process related genes, especially hematopoietic lineage-specific genes, associated with the responses to SOCS3 in HSCs.Downexpression of SOCS3 in HSCs or differentiated erythroid progenitor cells induced a transcriptional program enriched for erythroid development relative genes. Our results proved that SOCS3 down-expression induced lineage commitment towards erythroid progenitor cell fate by activation of erythroid-specific gene in HSCs and provided new insight into the mechanism of erythropoietic development.

Five hexanitratoneodymate-based rare earth complexes () were synthesized using a straightforward method. Purple plate crystals of were isolated and the crystal structure was determined by single-crystal X-ray diffraction with respect to the coordination mode of the nitrate anion to the central Nd(iii) ion. (: monoclinic system P21/c, a = 15.9460(3) Å, b = 10.2457(6) Å, c = 33.323(3) Å, β = 91.8108(17)°, V = 3109.11(11) Å(3), Z = 4). The central Nd(iii) ion is surrounded by six bidentate nitrate ligands, with a major trend towards high symmetry of the [Nd(NO3)6](3-) anion as an icosahedron. Thermal properties were determined from differential scanning calorimetry (DSC) combined with thermogravimetric analysis (TGA) tests. Complexes are found to be room temperature liquids, and their excitation and emission spectra were recorded. These complexes exhibit intense near-infrared (NIR) luminescence emission, which originates from interconfigurational f-f transitions (4)F3/2→(4)IJ multiplet (J = 9/2-13/2). These liquid Nd(iii) complexes are of interest as potential NIR luminescent soft materials with high thermal stability.

Glioma is the most common malignant intracranial tumors. Despite newly developed therapies, these treatments mainly target oncogenic signals, and unfortunately, fail to provide enough survival benefit in both human patients and mouse xenograft models, especially the first-generation therapies. Oridonin is purified from the Chinese herb Rabdosia rubescens and considered to exert extensive anti-cancer effects on human tumorigenesis. In this study, we systemically investigated the role of Oridonin in tumor growth and the underlying mechanisms in human glioma. We found that Oridonin inhibited cell proliferations in a dose- and time-dependent manner in both glioma U87 and U251 cells. Moreover, these anti-cancer effects were also confirmed in a mouse model bearing glioma. Furthermore, cell cycle arrest in S phase was observed in Oridonin-mediated growth inhibition by flow cytometry. Cell cycle arrest in S phase led to eventual cell apoptosis, as revealed by Hoechst 33342 staining and annexin V/PI double-staining. The cell apoptosis might be accomplished through a mitochondrial manner. In all, we were the first to our knowledge to report that Oridonin could exert anti-cancer effects on tumor growth in human glioma by inducing cell cycle arrest and eventual cell apoptosis. The identification of Oridonin as a critical mediator of glioma growth may potentiate Oridonin as a novel therapeutic strategies in glioma treatments.

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

Background: It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase., Materials and Methods: We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes' ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency., Results: The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline., Conclusion: These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.

Background: It has been demonstrated recently that α1,3-galactosidase from Bacteroides fragilis can efficiently convert human group B red blood cells (RBC) to group O cells. In addition, in vitro data indicated that the enzymatic conversion process did not affect the physiological or metabolic parameters of the RBC. The aim of this study was to investigate the lifespan of enzyme- treated RBC in vivo in the circulation., Materials and Methods: This was an experimental, randomised study. The rat was selected as the experimental subject because it expresses α-1,3galactosyl on its RBC. The efficiency of Galα1,3Gal epitope removal from RBC treated with α1,3-galactosidase was tested before the transfusion experiment to track the survival of RBC in the circulation. The animals were divided into three groups and injected via the tail vein with native, mock-treated or enzyme-treated RBC labelled with fluorescein isothiocyanate. The survival rates of the fluorescently labelled RBC were monitored by flow cytometry., Results: Flow cytometry showed that α-galactosidase (0.02 mg/mL for RBC with a haematocrit of 30%) efficiently removed Galα1,3Gal epitopes from rat erythrocytes, although small amounts of remaining Galα1,3Gal epitopes were still detected. The in vivo data demonstrated that the half-life of enzyme-treated RBC was a little shorter than that of native RBC. However, the 24-hour survival fractions of native, mock-treated and enzyme-treated RBC were virtually identical. Most importantly, the enzyme-treated RBC, like the native RBC, were still detectable 35 days after transfusion., Discussion: Our results indicate that α-glycosidase treatment had little effect on the in vivo survival kinetics of RBC. These data add further support to the feasibility of translating enzymatic conversion technology into clinical practice.

Background: Previous studies have suggested that reducing the positive charge of melittin could increase endosomal release activity and improve branched polyethylenimine (BPEI)-mediated transfection. AR-23 is a melittin-related peptide from Rana tagoi, which shows 81% sequence identity with melittin but has less positively-charged residues than melittin. The present study aimed to investigate the mechanistic and functional aspects of the interaction of AR-23 with mammalian cells and thus improve BPEI-mediated gene transfection., Methods: AR23 and two AR-23 analogs (AR-20 without positively-charged residues and AR-26 with the same positively-charged residues as melittin) were analyzed. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. Peptide-induced depolarization of cell membrane, the membrane-lytic activity of the peptides, and their potency with respect to enhancing the cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined., Results: The CD spectra results indicated that a positive charge in AR-23 played a crucial role in maintaining the α-helical conformation, whereas an extra positive charge could not increase α-helical formation. AR-23 displayed a similar depolarization ability to melittin. However, AR-23 showed a lower membrane lytic activity under physiological conditions and a higher lytic activity at endosomal pH than melittin and AR-26, which possess more positive charges. Compared to melittin and AR-26, AR-23, with a higher endosomal escaping activity, resulted in a higher enhancement of BPEI-mediated gene transfection, as well as the maintainance of a lower cytotoxicity., Conclusions: We suggest that AR-23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers., (Copyright © 2013 John Wiley & Sons, Ltd.)

The hexanitratolanthanate anion (La(NO(3))(6)(3-)) is an interesting symmetric anion suitable to construct the component of water-free rare-earth-metal ionic liquids. The syntheses and structural characterization of eleven lanthanum nitrate complexes, [C(n)mim](3)[La(NO(3))(6)] (n=1, 2, 4, 6, 8, 12, 14, 16, 18), including 1,3-dimethylimidazolium hexanitratolanthanate ([C(1)mim](3)[La(NO(3))(6)], 1), 1-ethyl-3-methylimidazolium hexanitratolanthanate ([C(2)mim](3)[La(NO(3))(6)], 2), 1-butyl-3-methylimidazolium hexanitratolanthanate ([C(4)mim](3)[La(NO(3))(6)], 3), 1-isobutyl-3-methylimidazolium hexanetratolanthanate ([isoC(4)mim](3)[La(NO(3))(6)], 4), 1-methyl-3-(3'-methylbutyl)imidazolium hexanitratolanthanate ([MC(4)mim](3)[La(NO(3))(6)], 5), 1-hexyl-3-methylimidazolium hexanitratolanthanate ([C(6)mim](3)[La(NO(3))(6)], 6), 1-methyl-3-octylimidazolium hexanitratolanthanate ([C(8)mim](3)[La(NO(3))(6)], 7), 1-dodecyl-3-methylimidazolium hexanitratolanthanate ([C(12)mim](3)[La(NO(3))(6)], 8), 1-methyl-3-tetradecylimidazolium hexanitratolanthanate ([C(14)mim](3)[La-(NO(3))(6)], 9), 1-hexadecyl-3-methylimid-azolium hexanitratolanthanum ([C(16)dmim](3)[La(NO(3))(6)], 10), and 1-methyl-3-octadecylimidazolium hexanitratolanthanate ([C(18)mim](3)[La(NO(3))(6)], 11) are reported. All new compounds were characterized by (1)H and (13)C NMR, and IR spectroscopy as well as elemental analysis. The crystal structure of compound 1 was determined by using single-crystal X-ray diffraction, giving the following crystallographic information: monoclinic; P2(1)/c; a=15.3170 (3), b=14.2340 (2), c=13.8954(2) Å; β=94.3453(15)°, V=3020.80(9) Å(3), Z=4, ρ=1.764 g cm(-3). The coordination polyhedron around the lanthanum ion is rationalized by six nitrate anions with twelve oxygen atoms. No hydrogen-bonding network or water molecule was found in 1. The thermodynamic stability of the new complexes was investigated by using thermogravimetric analysis (TGA). The water-free hexanitratolanthanate ionic liquids are thermal and moisture stable. Four complexes, namely complexes 8-11, were found to be ionic liquid crystals by differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). They all present smectic A liquid-crystalline phase., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.

Background: Melittin is a commonly used cell-penetrating peptide (CPP) for improving branched polyethylenimine (BPEI)-mediated gene transfection. However, its application is limited owing to the cytotoxicity generated by the lytic activity at neutral pH. In the present study, we report two truncated peptides from melittin and florae with improved transfection efficiency., Methods: Two truncated peptides consisting of 1-20 residues of melittin (MT20) and florae (FL20) were synthesized. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. The membrane-lytic activity of the peptides and their potency in enhancing cellular uptake of calcein were evaluated. The peptides and BPEI mixtures were mixed with plasmid DNA to prepare peptide/BPEI/DNA complexes. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined., Results: CD analysis and structure observation showed that the truncated peptides have α-helical conformation, which was necessary for penetrating activity. The truncated peptides exhibited several advantages than their parent peptides: (i) they showed higher hemolytic potency in acidic pH but lower lytic activity than their parent peptides in neutral pH; (ii) enhanced calcein efficiently release from both early and late endosome; (iii) they did not affect the DNA-binding affinity of BPEI and the physicochemical characteristics of BPEI/DNA complexes. Moreover, the peptides could increase BPEI-mediated transfection efficiency in different cell lines (293FT, B16F10 and CHO-K1) by simply mixing with BPEI, without causing cytotoxicity., Conclusions: The results obtained in the present study indicate that the truncated peptides with higher endosomal disrupting activity were better enhancers for increasing transfection efficiency., (Copyright © 2012 John Wiley & Sons, Ltd.)

A family of thermoresponsive cationic copolymers (TCPs) that contain branched PEI 25 K as the cationic segment and poly(MEO(2)MA-co-OEGMA(475)) as the thermosensitive block (TP) is prepared. The DNA binding capability, physicochemical properties, and biological performance of the TCPs are studied. All of these TCPs can condense DNA to form polyplexes with diameters of 150-300 nm and zeta potentials of 7-32 mV at N/P ratios between 12 and 36. The length of TP block is a key factor for shielding the positive surface charge of the polyplexes and protecting them against protein adsorption. TCPs with a higher TP content have a lower cytotoxicity while the best transfection performance is achieved by the TCPs with longest TP length, reaching a level of the intact PEI 25 K in the presence of serum., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The preparation and application of universal group O donor red blood cells (RBC) are a trend of future transfusion medicine. This article reviewed the technologies for producing universal RBC in recent years. One of them is modification of blood group antigens, which includes two basic methods. One of these two methods is enzymatic cleavage of the terminal immunodominant sugars from carbohydrate chains on the membrane of group A or/and group B RBC, in order to produce so-called enzyme-converted group O (ECO) RBC. ECO RBC have been produced from whole units of B RBC, which then survived normally when given to type A and O individuals in clinical trial. Because of the complexity of group A antigens, conversion of group A RBC (especially A1 RBC) to group O RBC is more difficult. Recently, a new bacterial glycosidase efficiently cleaving antigens on the surface of both A₁ and A₂ RBC has been obtained. Another method is pegylation, which camouflage the antigens on the surface of RBC with non-immunogenic molecules such as polyethylene glycol (PEG) in a non-specific way, to provide O, minor antigen negative phenotype RBC. The second technology is generating universal RBC from stem cells (such as hematopoietic stem cells, human embryonic stem cells) and human dermal fibroblasts, which will provide a new resource for blood supply. Great progress has been made, but a number of challenges still remain for using them in clinical transfusion, including scale-up, effectiveness and safety of prepared RBC. However, these researches will provide solutions for the problems in current transfusion, such as blood supply shortage, blood borne disease and emergency blood transfusion, and enhance the safety of clinical transfusions in the near future.

This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Aim: To clarify the therapeutic strategies and prognosis factors of primary clear cell carcinoma of the liver (PCCCL)., Methods: The clinical pathological data of 64 patients with PCCCL treated with hepatectomy in our hospital from January 2000 to January 2006 were analyzed retrospectively. The patients were divided into two groups to make treatment analysis: curative resection only (n = 40); and curative resection and postoperative chemotherapy with calcium folinate and tegafur (n = 24). Meanwhile, the PCCCL patients were subdivided into two subgroups on the basis of the proportion of clear cells in the tumor for pathological analysis. There were 36 cases in subgroup A for which the proportion of clear cells was more than 70%, and 28 cases in subgroup B for which the proportion was less or equal to 70%, comparing analysis of median survival time of the counterpart groups. Univariate and multivariate analyses were performed to examine factors that affected clinical prognosis, recurrence and metastasis., Results: Median survival period of the curative surgery group was 38 mo, while the counterpart was 41 mo. Median survival period for group A was 41 mo, while group B was 19 mo. The Kaplan-Meier method showed that capsule formation, preoperative liver function, hepatitis C virus infection, large vascular invasion and multiple tumor occurrences were related to disease-free survival. Cox regression analysis showed that the clear cell ratio, capsule formation, preoperative liver function and large vascular invasion were independent risk factors for overall survival., Conclusion: Postoperative chemotherapy has no obvious effect on survival of patients with PCCCL. Clear cell ratio, capsule formation, preoperative liver function, and vascular invasion were independent risk factors for prognosis.

Background and Objective: Epigenetic silencing of the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), is associated with the therapeutic response to methylating agents. This study was to assess the value of detecting the promoter methylation of MGMT gene in chemotherapy for glioma., Methods: Methylation-specific PCR (MSP) was employed to detect MGMT promoter CpG island methylation in 39 samples of glioma taken from surgery. Western blot and immunohistochemistry were used to detect protein expression. MTT were employed to detect the sensitivity of two glioma cell lines to alkylating agents, ACNU and TMZ. The Kaplan-Meier curve was adopted to estimate the overall survival according to the methylation status of the MGMT promoter., Results: Methylation of MGMT promoter CpG island was detectable in 46.2% of glioma tissues, but not in any normal tissues. The expression rate of MGMT protein was 61.5%. The status of MGMT methylation status was association with the protein level of MGMT (P<0.05). The MGMT gene was demethylated in glioma cell line SHG-44 following 5-Aza-CdR treatment; the expression of MGMT protein was restored and the resistance of SHG44 cells to alkylating agents was reversed. The overall survival was higher in patients with methylated MGMT promoter than in those with unmethylated MGMT promoter (P<0.05)., Conclusions: The status of MGMT promoter CpG island methylation is closely correlated to MGMT protein expression and sensitivity of cells to alkylating agents in glioma. Detection of the methylated sequences of MGMT may be used as a predictive factor for the treatment of glioma.

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.

The study was aimed to investigate the possibility of enhancing transfection efficiency of branched polyethylenimine (BPEI) in HeLa cells by hydrophobic tail of bee venom peptide (melittin). Hydrophobic tail of melittin was synthesized and its membrane permeable activity was evaluated by hemolysis test. The peptide was mixed with BPEI and the transfection efficiency was determined in HeLa cells by using green fluorescent protein gene (GFP) as a reporter gene. The cytotoxicity of the mixture was analyzed by MTT assay at 24 hours after transfection. The results indicated that the synthesized peptide had permeable activity leading to hemolysis in both neutral and acidic solution. At optimal condition, the peptide could significantly improve the transfection efficiency of BPEI and the cytotoxicity of the mixture was lower than BPEI itself. It is concluded that hydrophobic tail of melittin may be a potential enhancer to improve transfection efficiency mediated by cationic polymers in difficult to transfect cells.

Background: Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell., Methods: alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O., Results: The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe., Conclusion: ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

The widespread distribution of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the adult brain suggests its role in a broad range of brain functions. Here we show evidence supporting a physical interaction of PTEN with a region in the third intracellular loop (3L4F) of the serotonin 5-HT2C receptor (5-HT2cR, formerly 5-HT1c receptor) in cell cultures. PTEN limits agonist-induced phosphorylation of 5-HT2cR through its protein phosphatase activity. We showed the probable existence of PTEN:5-HT2cR complexes in putative dopaminergic neurons in the rat ventral tegmental area (VTA), a brain region in which virtually all abused drugs exert rewarding effects by activating its dopamine neurons. We synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F or the 5-HT2cR agonist Ro600175 suppressed the increased firing rate of VTA dopaminergic neurons induced by delta9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Using behavioral tests, we found that Tat-3L4F or Ro600175 blocks conditioned place preference of THC or nicotine, and that Ro600175, but not Tat-3L4F, produces anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggest a potential strategy for treating drug addiction with the Tat-3L4F peptide.

In order to study the possibility of xenotransfusion from porcine red blood cell (pRBC) to primate, the antigens on pRBC surface were modified to make it more compatible to primate sera. Porcine RBCs were subjected to both enzymatic removal of membrane alpha-Gal antigens with recombinant alpha-galactosidase (AGL) and covalent attachment of succinimid propionate-linked methoxypolyethyleneglycol (mPEG-SPA) to camouflage non-alphaGal antigens. The effects of double modifications were determinated by hemagglutination and clinical cross-match testing with rhesus sera. In vivo clearance rates and safety of modified pRBCs were measured after it was transfused into Rhesus monkey with or without immunosuppressant treatment. The validity of pRBC was detected in exsanguine Rhesus monkey model. The results showed that AGL could effectively remove alpha-Gal xenoantigens on pRBC membrane and reduce hemagglutination. The combination of mPEG modification with AGL treatment could significantly increased compatibility between pRBCs and Rhesus monkey sera. Modified pRBCs were detectable in Rhesus monkey blood at 12 hours after transfusion, and their survival time was 40 hours in the immunosuppressant-treated Rhesus monkey. In vivo survival rates of pRBCs were 38% in exsanguine Rhesus monkey at 8 hours after transfusion, and during that time, the hemoglobin and hematocrit of Rhesus monkey were maintained at the same level as before it lost blood. It is concluded that the modified pRBC can be safely transfused into Rhesus monkey and relieve the anemic symptom exsanguine Rhesus monkey. It suggested that pRBC can be hopefully used as a blood substitute for primate and human in the future.

The hippocampal dentate gyrus in the adult mammalian brain contains neural stem/progenitor cells (NS/PCs) capable of generating new neurons, i.e., neurogenesis. Most drugs of abuse examined to date decrease adult hippocampal neurogenesis, but the effects of cannabis (marijuana or cannabinoids) on hippocampal neurogenesis remain unknown. This study aimed at investigating the potential regulatory capacity of the potent synthetic cannabinoid HU210 on hippocampal neurogenesis and its possible correlation with behavioral change. We show that both embryonic and adult rat hippocampal NS/PCs are immunoreactive for CB1 cannabinoid receptors, indicating that cannabinoids could act on CB1 receptors to regulate neurogenesis. This hypothesis is supported by further findings that HU210 promotes proliferation, but not differentiation, of cultured embryonic hippocampal NS/PCs likely via a sequential activation of CB1 receptors, G(i/o) proteins, and ERK signaling. Chronic, but not acute, HU210 treatment promoted neurogenesis in the hippocampal dentate gyrus of adult rats and exerted anxiolytic- and antidepressant-like effects. X-irradiation of the hippocampus blocked both the neurogenic and behavioral effects of chronic HU210 treatment, suggesting that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects likely via promotion of hippocampal neurogenesis.

The mechanisms underlying brain seizure tolerance, a phenomenon in which brief periods of seizures protect brain against the lethal effects of subsequent sustained seizures, are poorly understood. Because brain seizure tolerance and brain ischemia tolerance likely share certain common mechanisms, the recent evidence that activation of extracellular regulated kinase (ERK) and p38 kinase pathways plays a critical role in ischemic preconditioning suggests that a similar mechanism may underlie brain seizure tolerance. We investigated the hypothesis in a rat kainic acid preparation of seizure preconditioning and tolerance, which was established by induction of one episode of priming epileptic status lasting for 20 min on the first day and another episode of sustained epileptic status lasting for 2 hr on the second day. We observed that acute seizures lead to a rapid activation of ERK and p38 in the hippocampal CA3 area, the brain region most susceptible to the lethal effects of epileptic status. Pretreatment with the ERK inhibitor PD98059 and the p38 inhibitor SB203580 selectively reduces seizure-elicited activation of ERK and p38, respectively, and significantly reduces priming seizure-induced protection of CA3 neurons. These findings indicate that, similar to brain ischemia tolerance, brain seizure tolerance also involves the ERK and p38 signaling pathways., (2005 Wiley-Liss, Inc.)

Background & Objective: Glioma is a common tumor in central nervous system with no specific clinical therapy. Its pathogenesis is unclear. This study was to clone tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction (PCR)-based subtractive hybridization, and to explore the molecular biological mechanism of tumorigenesis of glioma., Methods: mRNA was isolated from a sample of human glioma, and reversely transcribed into cDNA. PCR-based subtractive hybridization was used to clone tumor-related genes and tumor suppressor genes from it., Results: In tumor-related candidate gene group, phospho-protein enriched in astrocytes of 15 (PEA15) and homology of acid fibroblast growth factor (aFGF) were picked up. Whereas, in tumor suppressor gene group, interferon-induced protein 17 and ndr2 were picked up. ndr2 was widely expressed in normal brain tissue, but absent in glioma tissue., Conclusion: ndr2 gene is a candidate tumor suppressor gene, and may play a role in tumorigenesis of glioma.

Arsenic compounds (As(2)O(3 )or()As(4)S(4)) have been used successfully for the treatment of acute promyelocytic leukemia (APL) for quite a long time. It has been noticed that the sensitivity to apoptosis induced by As(2)O(3 )varies among various leukemia cells. It was reported by several groups that As(2)O(3) could induce apoptosis in APL-derived NB4 cells at concentrations of 0.5-1 mumol/l, whereas in other leukemia cells like K562, As(2)O(3) has no effects at the same concentration. K562 cells undergo apoptosis only when the concentration of As(2)O(3 )is greater than 2 mumol/l. Another arsenic compound, realgar (As(4)S(4)), a traditional Chinese mineral medicine, has been used to treat APL effectively and demonstrated to have lower toxicity than As(2)O(3). It would be interesting to know whether NB4 and K562 cells will show different sensitivity to realgar as well and if there is a difference, what is the cellular mechanism of it. In our present study, K562 cells were much less sensitive than NB4 cells to apoptosis induced by realgar. We confirm that the expression of bcl-x(L) is significantly higher in K562 cells than that in NB4 cells and is not downregulated upon realgar treatment. K562 cells become sensitive to realgar at clinically acceptable concentrations when bcl-x(L) expression level is downregulated by transfecting bcl-x(L) antisense RNA vector into the cells. Our results suggest that the increased bcl-x(L) expression in K562 cells contributes to its insensitivity to realgar-induced apoptosis., (Copyright (c) 2005 S. Karger AG, Basel)

Objective: To study the mechanism of hepatitis C virus (HCV) gene regulation and the inhibitory effect of antisense RNA on HCV gene expression in vitro., Methods: The hepatoblastoma cell line (HepG2) was co-transfected by recombinant plasmid of antisense RNA complementary to HCV 5' untranslated region (5'UTR)and HCV 5' UTR Directed luciferase (luc) gene expression recombinant plasmid. Meanwhile a reversed HCV 5'UTR recombinant plasmid which can not transcribe as antisense RNA in the cell and a recombinant plasmid in which the luc was regulated by simian virus 40 (sv40) 5'UTR were used as controls respectively. The level of luc gene expression was determined by an enzymatic assay., Results: The antisense RNA which directed to HCV 5'UTRcould obviously knock down the level of luc gene expression and the close-dependent inhibition of antisense RNA was observed at the same time. However the above inhibition was not shown in the cells co-transfected by reversed HCV 5'UTR recombinant plasmid and HCV 5'UTR directed luc gene expression recombinant plasmid. No reduction was observed in luc gene expression level in the cell co-transfected by both antisense RNA recombinant plasmid and SV40 5'UTR directed luc gene expression recombinant plasmid., Conclusion: HCV 5'UTR plays an important role in regulation of viral gene expression. The antisense RNA complementary to HCV 5'UTR could effectively inhibit the gene expression regulated by HCV 5'UTR in vitro.

Aim: To find out why mPEG modification of donor's lymphocytes can attenuate the occurrence of graft versus host disease(GVHD), but not affect the hemopoietic reconstitution of stem/progenitor cells after transplanting the mPEG-modified mononuclear cells from human cord blood into the SCID mice., Methods: The followings were observed: (1) Changes of CD4(+) and CD8(+) T cells and the ratio of CD4(+)/CD8(+) T cells were examined by flow cytometry before and after mononuclear cells from human cord blood were modified with mPEG. (2) The difference in forming the CFU-GM in-vitro between the mPEG modified-stem/progenitor cell group and non-modified cell group was observed. (3) The time of appearance of GVHD and the survival of the SCID mice were observed after the pre- and post-modification mononuclear cells were transplanted. (4) The number of humanized CD45(+) cells in the mouse's bone marrow was detected about 7 weeks after transplantation., Results: (1) mPEG nearly completely covered up the CD4 and CD8 antigens on T cells, while the number of CFU-GM did not show any obvious change between the modified and non-modified cell groups. (2) GVHD appeared later in the modified mononuclear cell group than in the non-modified group, and the survival rate was elevated in the modified group than in the non-modified group. (3) Humanized CD45 cells were found in mouse's bone marrow at the 47th day after transplantation of both mPEG-modified and non-modified mononuclear cells., Conclusion: After CD4 and CD8 antigens were covered up with mPEG, the graft's immune response against host was weakened, but the proliferation and differentiation of transplanted hemopoietic stem/progenitor cells were not affected.

Background & Objective: Ndr2 (N-myc down stream regulator) gene in human is a new gene cloned with the human adult whole brain cDNA as template in 1999, which accession number is AF159092 in GenBank. Locating backward position of the N-myc gene in human chromosome, this gene was named Ndr2 gene. The previous experimental results showed Ndr2 gene probably is a tumor suppressor gene. To research the function of Ndr2 gene, the authors cloned the genomic sequence of ndr2 from mouse., Methods: To clone Ndr2 genomic sequence by reverse transcription-polymerase chain reaction(RT-PCR) with the mouse genome library as template; automatic sequencing was performed using 310 Genetic Analyzer; homogeneous analysis was made using GenBank BLAST; open reading fragment(ORF) analysis was made using PC Gene and ORF Finder; domain analysis was made using ProDom system., Results: A fragment (about 3310bp,identified by agarose gel electrophoresis) was obtained using RT-PCR with the mouse genome library as template. The fragment was cloned in pMD18-T vector. BLAST analysis showed that the sequence was highly homogeneous (with the homogeneity rate of 91.4%) with Ndr2 gene in human and non-homogeneous with genomic sequence database in mouse. ORF analysis showed that there was a complete coding region in it, which including 8 extrons and 7 introns; it can interpret a protein containing about 200 amino acid residuals. ProDom analysis showed there was a domain like acyl carrier protein(ACP) in it., Conclusion: The authors cloned Ndr2 gene in mouse and proved that the sequence is a new genome sequence in mouse genomic sequence database. At present, the genome sequence has been submitted to GenBank(the accession number: AY151387).

Oxysterol binding protein (OSBP) is a regulator of oxysteroid metabolism. To investigate the function and the structure-function relationship of OSBP, the recombinant vector OSBP PH-pRSET-A was transformed into E.coli JM109(DE3), and the strain highly expressing soluble 6His-OSBP PH domain in minimal medium were obtained. The fusion protein was purified by Ni(2 )-NTA agarose beads. The secondary structure of the purified 6His-OSBP PH domain fusion protein was analysed by circular dichronism. The results indicated the PH domain was composed of alpha-helix 7.2%, beta-pleated sheets 71.1% and radom coil 21.7%.