Mammalian integrated stress responses in stressed organelles and their functions.

The integrated stress response (ISR) triggered in response to various cellular stress enables mammalian cells to effectively cope with diverse stressful conditions while maintaining their normal functions. Four kinases (PERK, PKR, GCN2, and HRI) of ISR regulate ISR signaling and intracellular protein translation via mediating the phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51. Early ISR creates an opportunity for cells to repair themselves and restore homeostasis. This effect, however, is reversed in the late stages of ISR. Currently, some studies have shown the non-negligible impact of ISR on diseases such as ischemic diseases, cognitive impairment, metabolic syndrome, cancer, vanishing white matter, etc. Hence, artificial regulation of ISR and its signaling with ISR modulators becomes a promising therapeutic strategy for relieving disease symptoms and improving clinical outcomes. Here, we provide an overview of the essential mechanisms of ISR and describe the ISR-related pathways in organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. Meanwhile, the regulatory effects of ISR modulators and their potential application in various diseases are also enumerated.

Facile Synthesis of Optically Active and Magnetic Nanoparticles Carrying Helical Poly(phenyl isocyanide) Arms and Their Application in Enantioselective Crystallization.

In this paper, the facile synthesis of hybrid Fe3 O4 magnetic nanoparticles carrying helical poly(phenyl isocyanide) (PPI) arms via both "grafting from" and "grafting onto" strategies is reported. First, alkyne-Pd(II) catalysts are anchored onto the surface of the Fe3 O4 magnetic nanoparticle, which promote the polymerization of enantiopure phenyl isocyanide, affording the expected hybrid magnetic nanoparticle with Fe3 O4 in core and helical PPI as arms. The nanoparticle also exhibits highly optical activity due to the excess of one-handed helicity of the PPI arms. Moreover, the hybrid magnetic nanoparticle can be alternatively synthesized via "grafting onto" strategy. A triethoxysilanyl-terminated single handed helical PPI bearing l-alanine ester pendants is prepared and grafted onto the surface of Fe3 O4 nanoparticle. The generated hybrid magnetic nanoparticles show both magnetic character and optical activity. Taking advantage of these properties, they can be used in enantioselective crystallization of racemic threonine. The enantiomeric excess (ee) of the induced crystals is up to 93%. Moreover, the nanoparticles can be facilely recovered and recycle used for at least four times in enantioselective crystallization without significantly loss of its enantioselectivity.

[Relationship between the gene polymorphism in fibroblast growth factor-10 and susceptibility to chronic obstructive pulmonary disease 220 cases].

OBJECTIVE: Fibroblast growth factor 10 (FGF 10) signaling pathway is crucial to lung development and epithelial reconstruction. The aim of this study was to investigate the relationship between gene polymorphisms in FGF 10 and susceptibility to chronic obstructive pulmonary disease (COPD) in a Han population of North China. METHODS: The subjects included 220 patients with COPD (COPD group) and 285 healthy controls (control group). The COPD patients, admitted to our hospital from June 2007 to May 2012 because of acute exacerbation, included 142 males and 78 females, aging from 43 to 93 years [mean (74 ± 10)]. The control group included 183 males and 102 females, aging from 44 to 97 years [mean (72 ± 9)]. According to results of lung function testing, patients with COPD were divided into mild (8 cases), moderate (62 cases), and severe (48 cases) and very severe groups (102 cases). The genotype frequencies of FGF 10 gene single nucleotide polymorphisms (rs10473352, rs16873956, rs2973644, rs1011814, rs10402070 and rs723166) were genotyped by RFLP PCR-restriction fragment length polymorphism method and micro-sequencing (SnaPshot) technology assay. Chi-square test was used to perform the Hardy-Weinberg equilibrium test. Unconditional logistic of regression model was used to calculate odds ratio (OR) and 95%CI. The 2 groups were compared using t-test. RESULTS: The rs2973644 locus AA, GA and GG genotype frequencies in the COPD group and the control group were 108/220, 92/220, 20/220 and 167/285, 104/285, 14/285, respectively; while the frequencies of allele A and G were 308/440, 132/440 and 438/570, 132/570, respectively. The rs10473352 locus TT, TC and CC genotype frequencies in the COPD group and the control group were 142/220, 72/220, 6/220 and 202/285, 82/285, 1/285, respectively, the differences being statistically significant (χ(2) value were 6.021-6.213, P < 0.05). The rs10473352 allele T and C frequencies were 356/440, 84/440 and 486/570, 84/570, respectively, the differences being not statistically significant (χ(2) = 3.395, P > 0.05). The rs1011814 locus TT, TC and CC frequencies in mild and moderate compared with severe and very severe COPD disease were 16/68, 39/68, 13/68 and 29/150, 67/150, 54/150, respectively; while its allele T, C frequencies were 71/136, 65/136 and 125/300, 175/300, respectively; the differences being statistically significant (χ(2) values were 6.287 and 4.200, all P < 0.05). The remaining 5 tSNP (rs10473352, rs16873956, rs2973644, rs10402070 and rs723166) genotype distribution and allele frequencies were not significantly different (OR value were 0.606-1.357, P > 0.05). CONCLUSIONS: FGF 10 gene SNP sites rs2973644 and rs10473352 polymorphisms may be associated with susceptibility to COPD in Han population of North China. The SNP in rs1011814 may be associated with severity of COPD.

[Establishment of a human nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP and screening of drug-resistant genes].

BACKGROUND & OBJECTIVE: Chemotherapy constitutes one of the chief supplementary methods in the treatment of nasopharyngeal carcinoma (NPC). However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to screen drug-resistant associated genes so as to identify potential molecular targets. This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE2, and to screen human nasopharyngeal carcinoma drug-resistant genes by a new strategy based on improved subtractive hybridization. METHODS: The drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay. Fluorescence activated cell analysis(FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Cell growth curve, doubling time, and cell morphology were measured and observed. The drug-resistant genes were screened by a new strategy of PCR-based subtractive hybridization. Sequencing and blast analysis were performed after the differentially expressed genes had been verified by reverse dot blotting. The result was further confirmed by RT-PCR. RESULTS: The resistance indexes of CNE2/DDP to cisplatin (DDP), 5-fluorouracil (5-FU), and vincristine (VCR) were 27.9, 227.9, and 55.5, respectively, indicating its multi-drug resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lower in CNE2/DDP cells than in CNE2 cells (12.98 vs. 243.62). The CNE2/DDP cells appeared smaller, more regularly round, and longer doubling time (26 hours vs. 19 hours) than CNE2 cells. Six differentially expressed sequences were discovered using improved subtractive hybridization; all of them were found to be homologous to known genes after sequencing analysis. Three of them were highly expressed in CNE2/DDP cells. Among them, one sequence, which encodes a 79 amino acid protein,known as DC13 protein (DC13), was a function unknown gene which has certain relationship with malignancy. The other two sequences were ubiquitin C gene and NADH dehydrogenase subunit 2 (ND2) gene, respectively. The other three of the six sequences, whose expression were inhibited in CNE2/DDP cells, were cytochrome C oxidase subunit I(COX1), ribosomal protein L27(RPL27),and ribosomal protein S27 (RPS27) genes, respectively. CONCLUSION: A drug- resistant cell line CNE2/DDP, which showed a typical resistant phenotype to anti-cancer drugs was established. The PCR-based improved subtractive hybridization is an effective approach to identify differentially expressed genes. Many genes, both known and unknown, might contribute to the existence of drug-resistant phenotype, through increasing or decreasing their expression.

[Soluble expression of peptide containing MUC1/Y-specific epitope in Escherichia coli and preparation of the antibody].

MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.