Abstract:Nerve growth factor, a kind of neurotrophic factor, plays an important role in neuronal development, differentiation, survival and neurogenesis, and is considered as a link between neuroendocrine system and immune system in asthma attack. The possible mechanism of effects of nerve growth factor on asthma is as follows: (1) nerve growth factor changes airway innervation, and facilitates the synthesis and release of neurotransmitter in nerve terminal, which will contribute to airway remodeling; (2) nerve growth factor induces eosinophils aggregation, proliferation and releasing inflammatory factor, which will lead to the abnormality of immunologic response; (3) nerve growth factor triggers the redundancy of adrenal medullary cells, which results in adrenal medullary cell to nerve cell transition, and then the impairment of chromaffin cell endocrine secretion function. As a result, the concentrations of adrenaline in circulation are not competent to relieve the bronchoconstriction in asthmatic attack.

Abstract:With the advent of post-genomic era, identification of protein-protein interaction has become another hot spot of protein research and promoted the invention, development and complement of relative techniques. Major large-scale high-throughput methods, such as two-hybrid system, bacteriophage display, mass spectrometry, protein microchips and bioinformatics have provided a global view of protein-protein interaction, and are hopeful to play an important role in proteomics. Each method may have its specific strengths and weaknesses as well as differences in coverage, to some extent, the data based on these methods can complete each other. Here, recent progress of these large-scale high-throughput methods and their applications for studying protein-protein interaction are reviewed.

Abstract:Natural killer (NK) cells play an important role in the immune response to viral infection and tumors.The ability of NK cells depends on the integrated signals across the array of stimulatory and inhibitory receptors engaged upon interaction with target cell surface ligands. Some stimulators, including viruses, tumor cells and hot shock, could promote the expression of NKG2 receptors and their ligands via activating certain transcription factors which are capable of up-regulating NKG2 promoters' activity. Meanwhile, epigenetic mechanisms including DNA methylation and histone posttranslational modifications are also critical to expression of NKG2 receptors and their ligands and may control the clonally distribution of some NK cell receptors. Investigating the epigenetic mechanisms of NK cell receptors and their ligands might helpful to the rational design of therapy against infection, inflammation, cancer or autoimmune diseases.

Abstract:Obestatin was a newly found ghrelin-associated peptide(GAP), which could be bound to the orphan G protein-coupled receptor GPR39. Obestatin suppressed food intake, inhibited gastrointestinal function, and decreased body-weight gain which was regarded as the biological equally matched material or Yin and Yang activated polypeptide. However, recent reports indicated that obestatin could not specifically bind to GRP39 receptor, and could not alter ghrelin-induced biological functions. According to the reports above, researches related to obestatin and GPR39 were reviewed in the article.

Abstract:Generation, aggregation, and deposition of amyloid β-peptide (Aβ) in the brains of Alzheimer's disease (AD) patients are inherent pathological features of AD. Cleavage of the amyloid precursor protein (APP) by the β-secretase (BACE) is the first step in the generation of Aβ. Inhibition of BACE activity is a promising therapeutic strategy for the treatment of AD. Therefore, understanding the structure of BACE is very important. Using blue native gel electrophoresis it has been found that BACE exists as a homodimer. However, it is currently unknown whether in living cells BACE forms homodimer. To address this issue, the dimerization of BACE in intact living cells was monitored by using confocal microscopy and acceptor photobleaching FRET. Using bioengineering technique, fluorescence proteins (FPs)-tagged BACEs (BACE/FPs) was constructed. They are cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) tagged full length BACE (BACE-FL) and truncated BACE (BACE-NT). The CFP- and YFP- tagged BACE-FL (BACE-FL/FPs) or CFP- and YFP- tagged BACE-NT (BACE-NT/FPs) plasmids were transfected into HeLa cells, and the expression and location of BACE-FL and BACE-NT were observed by confocal microscopy. The dimerization of BACE was evaluated by the FRET between CFP- and YFP- tagged BACE which was detected by acceptor photobleaching method. The results show that the BACE-FL can be transported to the Golgi apparus, plasma membrane and endosomes. However, the BACE-NT is found in the ER 24 hours after transfection. The FRET efficiency of BACE-FL/FPs group is (12.90±0.73)%, significantly higher than that of control group (3.32±0.55%) (P < 0.05). The FRET efficiency of BACE-NT/FPs group is (3.80±0.74)%, which is not statistically different from that of control group. These results indicat that the BACE-FL exists as a dimmer in living cells, but the BACE-NT exists as a monomer, suggesting the transmembrane and C-terminus regions are important for normal transport and localization, and is necessary for the dimerization of BACE.

Abstract:Ring dermoid of cornea (RDC) is an autosomal dominant disorder of cornea. The previous study identified a G185A mutation of PITX2 gene in a Chinese family with RDC. To investigate the pathological mechanism of PITX2 R62H mutation, a PITX2 prokaryotic expression plasmid were constructed and GST-PITX2 fusion protein were purified. EMSA was further conducted. The DNA-banding ability of PITX2 R62H was similar to that of the wild type PITX2 were found. The cell lines stably expressed PITX2 was also generated, and cell cycle were analyzed by flow cytometry, and the expression of β-catenin and Cyclin D1 were detected by quantitative Real-time PCR. The results showed that proliferating ability of cells expressing PITX2 R62H was lower than that of cells expressing PITX2 WT, as well as β-catenin and Cyclin D1 mRNA level. These findings revealed that PITX2 R62H mutation affected the Wnt/β-catenin→PITX2 pathway, promoted the genes expressing abnormally, and led to abnormal cell proliferation and the formation of RDC, which may play an important role in pathogenesis of RDC.

Abstract:Plant programmed cell death (PCD) plays an important role in plant growth and development as well as defensive response against biotic and abiotic stresses. Fumonisin B₁ (FB₁) is a fungi toxin, which is a competitive inhibitor of ceramide synthase in de novo sphingolipid biosynthesis. FB₁ can induce PCD in both animal and plant cells. To dissect this pathway, a genetic screen for fumonisin B₁ resistant (fbr) mutants, and identified 11 fbr mutants was carried out. Genetic analysis showed that these 11 mutants belonged to 9 complementary groups or genetic loci. Here a detailed phenotypic analysis of fbr136 was reported. In addition to the resistance to FB₁, fbr136 also showed resistance other PCD-inducing compounds, including H₂O₂ and paraquat. Furthermore, FB₁-induced PR1 expression was reduced in fbr136, suggesting that a PCD pathway is likely impaired in the mutant. When stained with nitroblue tetrazolium (NBT), fbr136 showed a reduced accumulation of reactive oxygen species (ROS) induced by FB₁. The fbr136 mutation was roughly mapped onto chromosome Ⅲ, where no other fbr mutants have been previously identified. It is proposed that FBR136 may act as an important regulator in a sphingolipid-mediated PCD pathway, involved in the generation of ROS.

Abstract:Chronic stress can induce hippocampus injury such as neuron loss, dendrite atrophy, but its mechanism and molecular basis remain unclear up to now. To understand the molecular mechanism on protein level and find the crucial proteins which correlated with chronic stress-induced injury, two-dimensional electrophoresis was applied to separate the hippocampal total proteins of control group and restraint stressed rats, then the differential expressed proteins were detected by image analysis and identified by matrix assisted laser desorption /ionization time of flight mass spectrometry (MALDI-TOF-MS) as well as database searching. Moreover, the 2-DE results were verified on the mRNA level by semi-quantitative RT-PCR. The hippocampal 2-DE map with high resolution and good reproducibility of control and stress group rats were obtained. Fourteen differentially expressed protein spots were detected and eleven proteins were successfully identified, most of these proteins were involved in the process of energy metabolism and signal transduction. These results provide a clue for elucidating the mechanism of chronic stress-induced hippocampal injury and are useful for elevating the adaptability to stress.

Abstract:Cell division is one of the key roles in cell development, cell differentiation, embryogenesis and recovery of tissues. As a frequent stimulus, stress (or strain) plays an important role in the differentiation of the cells, morphology generation and function development of tissues. However, this stimulus was not studied enough. A uniaxial static stretch device was used to investigate the influence of a uniaxial stress on stress fibers and spindle orientation of cultured Murine osteoblast line (MC3T3). The cells were seeded for 24 h on a thin rectangular silicon membrane in the uniaxial device before the static uniaxial stretch was applied to them. Five groups of the cells experiencing 4% strain for 48 h, 8% strain for 6 h, 8% strain for 12 h, 8% strain for 24 h, and 8% strain for 48 h were observed. After the stretching, the cells were incubated in fluorescent rhodamine-conjugated phalloidin and DAPI staining solution for 30 min. The stress fiber alignment and the cell division directions of the cells were recorded by CCD camera with an inverted fluorescent microscope (Olympus 71X) equipped with a green filter (554 nm). In fact, the cells that were growing individually were counted because the interaction between cells may influence the cell alignment and the mitosis orientation. These cells were divided into three groups, 0°～30°, 30°～60°, 60°～90°. The cells in each group were counted, then the distribution of the cell division directions were plotted in the circular graphs. It was showed that the morphology of the cells was regulated by 4% and 8% static uniaxial strain. Within 48 h, the uniaxial stress induced the stress fibers' alignment parallel to the stress direction while a random distribution of the long axial of the cells was seen in the control group. 49%, 43%, 54%, 54%, 62% of the cells under the five experimental conditions fall into the group Ⅰ(0°～30°) according to the angles between the stretch directions and the stress fibers alignments. And, there are 50%, 48%, 56%, 53%, and 62% of the cells under the five stretch conditions in group Ⅰ(0°～30°) according to the angles between the stretch and the cell division directions. Further statistic analysis indicates that the stress fibers' orientation and mitosis orientation show high relativity. The relativity was very high even 48 h after the stretch stimulus. These results suggest that the mechanical environment of the cells may play a role in the cell division orientation. The effect of cycle stress on cells mitosis orientation is worth further study.

Abstract:The standard protocol for generating mutant mice from heterozygous targeted ES cells currently is time-consuming and needs two breeding steps and, in some cases, risks the failure of germline transmission. In contrast, the tetraploid embryo complementation is a time-saving single step procedure escaping from the chimera mouse. Hybrid ES cell lines always have higher generation frequency because of their genetic heterozygosities, which also limit their application. However, inbred ES cell lines often fail to generate ES mice. Judged by microsatellite DNA marker analysis, the completely ES cell derived mice (ES mice) were efficiently produced with two inbred ES cell lines (SCR012 and CJ7) by microinjecting the ES cells into tetraploid blastocysts generated by electrofusion. As a result, after electrofusion, about (93.01±1.37)% of 2-cell stage embryos fused and (82.49±2.08)% of them developed to blastocysts after 48 h in vitro culture. The efficiency of generating newborn ES mice of wild type SCR012 cells was higher (8.3%) than that of CJ7 (2.7%). In the meanwhile, adult and fertile mutant ES mice with two gene knock-out ES cells-18 KO (derived from CJ7) and F8 KO (derived from SCR012) were generated. Interestingly, the generation frequency of 18 KO ES mice was even higher (8.1%) than wild-type CJ7 ES mice, indicating that the development potential among ES cell clones was significantly different from each other and the decline of development potential was not equal among ES cell clones during ES cell passage in vitro culture. The F8 KO mice had indistinguishable bleeding phenotype, which is identical to that of mice derived from breeding of chimeric mice. These results suggested that this technique can be used as a powerful approach to produced gene targeted mice efficiently.

Abstract:Six gene segments, PB1, PB2,PA, NP, M and NS, were fully synthesized which derived from the master donor virus(MDV), cold-adapted(ca)，temperature sensitive(ts), live attenuated influenza virus strain A/Ann Arbor/6/60(MDV-A). Meanwhile, five amino acid substitutions (PB1-391E, 581G, 661T, PB2-265S, NP-34G) were artificially altered by human intervention. HA and NA fragments derived from the 2006～2007 circulating strain A/New Caledonia/20/99 (H1N1). Eight fragments were ligated with modified pAD3000 for rescue plasmid construction. Eight transcription/expression plasmids were named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, respectively. The COS-1 cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the cDNA of the HA and NA segments of the strain A/New Caledonia/20/99 (H1N1), the results showed that cold-adapted, attenuated reassortant influenza A virus was rescued successfully. Titers of a reassorted influenza A virus in embryonated chicken eggs ranged from 1∶2⁹ to 1∶2¹⁰. The rescue system of six internal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted, live attenuated human influenza virus.

Abstract:Thyroid function ultimately depends on appropriate iodine supply to the gland. Thyroid hormone deiodination is an intrinsic component of the thyroid hormone homeostasis. Type Ⅰ iodothyronine deiodinase (D1) plays an important role in thyroid hormone metabolism and has close relationship with thyroid function. Based on successfully establishing animal models of iodine deficiency and iodine excess in Babl/c mice (Babl/c mice were randomly divided into five groups: low iodine (LI), normal iodine (NI), five-fold iodine (5HI) , ten-fold iodine (10HI) and fifty-fold iodine (50HI) group. Three months and six months after admistration, they were sacrificed and thyroids were excised), the expression level of D1 mRNA were examined by using real time quantitative PCR method. D1 activity was analyzed by ¹²⁵I-rT₃ as substrate combined with ion-exchange chromatography. The thyroid hormone was measured with radioimmunoassay method. The data revealed that in the case of iodine deficiency, both D1 mRNA expression and D1 activity was greatly increased(compared with NI groups, P < 0.01). T₃ and T₄ in thyroid tissue was significantly decreased, but T₃ / T₄ was increased (P < 0.01). On the other hand, when faced to iodine excess, D1 mRNA expression was reduced (There was a tendency of decreasing in D1 mRNA expression in all HI groups compared with NI group, significant difference was found in 5HI and 50HI at 6 months ), while there was no remarkable effect on D1 activity. The thyroid hormone assay showed that T₃ / T₄ was decreased. In conclusion, D1 is a critical factor in the regulation mechanism of thyroid function. The up-regulated mRNA expression and activity under the state of iodine deficiency will improve the conversion of T₄ to T₃ to maintain the normal thyroid function. The inhibition of D1 expression induced by iodine excess may be taken as an effective way to protect organism from impairment caused by too much T₃.

Abstract:Glucose-6-phosphate dehydrogenase (G6PD) derives from the expression of the house-keeping gene G6PD. Recent studies have indicated that G6PD is related to tumor genesis, growth, clinical phenotype, therapy, and prognosis. To elucidate the relationship between G6PD and cancer, three siRNA sequences and one negative control sequence were designed based on the 3' noncoding region of the human G6PD gene. Two complementary single-strand DNA (sense and antisense) were designed and synthesized based on siRNA sequences. The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-U6.2/Lenti. One siRNA with higher interference efficiency than the other two was found after siRNA plasmid transfecting human skin A375 melanoma cells. After lentivirus particle packaging and virus production, the A375 cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain. Western blotting showed that the endogenous G6PD in the stable A375 cell strain was 0.257 ± 0.074, which was 11.17% of G6PD expression (2.301 ± 0.286) in wild type A375 cells. The final siRNA interference efficiency in this stable cell strain was 88.83%. The G6PD activity of A375-G6PDΔ was 21.53% of A375-WT. Further study showed that A375-G6PDΔ doubling generation time prolonged, and its proliferation was greatly inhibited and the cloning efficiency lowered 25%(P < 0.05), compared with A375-WT cell. FCM analysis indicated that apoptosis cell in A375-G6PDΔ was 2.86 times as that of A375-WT(P < 0.01) with 33.8 % increase of SPF(P < 0.05), 59.7 % raise of PI(P < 0.01), and 27.7 % decrease of G0/G1 phase(P < 0.01). Furthermore, apoptosis-associated protein check showed that Caspase-3 was 2.86 times as that of A375-WT(P < 0.01) with 54.7% descend of P53(P < 0.01). It is proposed that G6PD can maintain the growth and proliferation of A375 cell. G6PD deficiency probably restrains the change proceeding of G2/M phase to G0/G1 phase in A375Δ cell cycle through down-regulation P53 expression and up-regulation Caspase-3 expression. The role and mechanism of G6PD in cell growth, proliferation, and differentiation of tumor cells need to be further investigated.

Abstract:Little is known about the role of mannan-binding lectin (MBL) in adaptive immune responses although it is believed to be of importance in innate immunity. The interaction of MBL with Raji cells, THP1/CD14 cells, Jurket cells and human red blood cells (HRBCs) has been investigated by ELISA, and the characteristics of MBL binding to Raji cells by FACS. The results showed that MBL could bind to Raji cells, THP1/CD14 cells and Jurket cells except HRBCs, and the binding was dose-dependent. Furthermore, the binding of MBL to Raji cells was evident in a Ca²⁺-dependent manner, which was partially inhibited by some saccharides (mannose, glucose, or N-acetylglucosamine), C1q and anti-C1qR mAb. Similarly, it was also incompletely inhibited by recombinant human MBL carbohydrate recognition domains (rhMBL-CRD) protein and recombinant human MBL collagen-like region (rhMBL-CLR) protein, respectively, but completely inhibited by both. These data indicated that Raji cells express Ca²⁺-dependent and sugar-sensitive CLR-specific and CRD-specific MBL receptors, of which, the former was also shared with C1q. Further investigation showed that MBL could significantly inhibit Raji cell proliferation directly at higher concentrations (10～50 mg/L) in a dose-dependent manner, suggesting that MBL might also play certain roles in adaptive immune responses.

Abstract:Japanese encephalitis virus (JEV) (family Flaviviridae, genus Flavivirus) is an arbovirus of public health importance. The envelope glycoprotein of JEV is associated with viral attachment and fusion with host cell, determine the virus′s hemagglutination ability, cellular tropism, viral virulence, and induction of protective immune response. The domain Ⅲ of envelope protein (E protein) is an important region in inducing neutralizing antibodies against JEV. In order to study the antigenic structure of domain Ⅲ on E protein, domain Ⅲ of the envelope protein was expressed by fusion with GST in a pGEX-6p-1 vector. Western blot demonstrated that expressed fusion protein could be recognized by anti-JEV sera. To map the antigenic epitope of this region, a set of 14 partially overlapping short peptides spanning the domain Ⅲ were designed and expressed in fusing with GST. Then Western blot and ELISA reactivity of these short peptide fusion proteins to anti-JEV sera were surveyed, respectively. Four linear antigenic epitopes, E39 (³⁰⁵TEKFSFAKNPVDTGHG³²⁰), E45-1 (³⁵⁵VTVNPFVATSSA³⁶⁶), E48-1 (³⁷⁷PFGDSYIV³⁸⁴) and E49 (³⁸⁵VGRGDKQINHHWHKAG⁴⁰⁰) were identified. Immunization of mice with epitope fusion proteins revealed that all four proteins could elicit short peptide specific antibody. And in vitro neutralization test verified that E39 was linear neutralizing epitope. This result provides important basis for further structural and functional study of domain Ⅲ of JEV envelope protein, and development of diagnostic techniques and vaccines.

Abstract:To screen for serum biomarkers for lung squamous carcinoma, two-dimensional gel electrophoresis (2-DE) was performed to separate serum proteins from healthy individuals and stage 1 lung squamous carcinoma(LSC) patients, respectively. PDquest software was used to analyze 2-DE images, and the differential serum protein spots between the healthy individuals and LSC patients were identified by ESI-Q-TOF MS/MS. Then Western blot and immunohistochemistry were used to detect the expression levels of haptoglobin-2(HP-2), one of the differential proteins, in the sera and tumor tissues in the patients with LSC, respectively. 2-DE maps of serum proteins from healthy individuals and stage 1 LSC patients were established. Ten differential serum protein spots were detected, four proteins of which were identified by MS/MS. Western blot showed that the serum level of HP-2 in the LSC patients was significantly higher than that in healthy individuals, but was not associated with LSC staging. Immunohistochemistry showed that the expression level of HP-2 in the LSC tissues was significantly higher than that in the normal bronchial epithelial tissues adjacent to tumors. The results indicated that serum HP-2 protein is a candidate biomarker for LSC, and might be useful for diagnosis of LSC. Up-regulation of HP-2 in the LSC tissues may contribute to the high serum level of HP-2 in the patients.