Abstract:Protein is one of major bio-functional performers. As one of several crucial proteomic research approaches, protein microarray has these following advantages: high-throughput, high sensitivity, quick detection and so on. Meanwhile, there are some critical factors that are important to the further development of protein microarray technology, for example, how to express and purify proteins for the research of protein microarray, how to immobilize proteins onto the substrate and keep the bio-function of proteins immobilized. Nano-biotechnology and cell-free expression system have been used to fabricate protein microarray by the way of immobilizing target genes onto the substrate and directly expressing corresponding proteins, which provides a new strategy to fabricate more complicated microarray. The stragegy and its progress were summarized——fabrication of protein microarray based on DNA, including immobilization of target genes, cell-free expression to proteins, immobilization of renascence proteins, advantages and drawbacks of the methods of protein chip fabrication etc.

Abstract:Aminoacyl-tRNA synthetase catalyzing the first reaction of protein biosynthesis. In mammalian cells, eight aminoacyl-tRNA synthetases (aaRSs) and three auxiliary protein factors form a macromolecular aminoacyl-tRNA synthetases complex (aaRS complex). The three nonsynthetase protein factors, namely, p43, p38, and p18 were found to be involved in many other important life activities besides their roles in the complex. The auxiliary factor p43 was the precursor of endothelial monocyte activating polypeptideⅡ(EMAPⅡ), which involved in angiogenesis and apoptosis. The auxiliary factor p38 was crucial for the development of lung, and its abnormal accumulation in neuron would be related to the Parkinson’s disease. The auxiliary factor p38 and p18 could promote the repair of DNA damage via different pathways in a highly organized way. All these breakthroughs enhance our understanding about the interaction between the aaRS complex and the macromolecular signaling network and promote the studies on this field.

Abstract:Songbirds and humans both rely critically on auditory feedback for learning and maintaining accurate vocalizations. During vocal learning, juvenile songbirds learn to sing by using auditory feedback to match their variable song patterns to a memorized tutor model. During song maintaining, adult songbirds monitor the integrality and accuracy of its song on-line according to auditory feedback. This form of learning requires auditory-vocal integration. The output of songs and the information of auditory feedback are integrated in some areas of the bird?蒺s brain, and then direct next vocal activity. In recent years, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) has been demonstrated that it is necessary for feedback-dependent song decrystallization, and LMAN neurons exhibit highly selective responses to auditory presentation of the bird?蒺s own song (BOS). Moreover, the discovery of mirror neurons in the high vocal centre (HVC) also provides a significant clue for further study in songbirds. Here the progress of auditory feedback and the plasticity of vocal learning in songbirds are reviewed in recent years.

Abstract:Exocyst complex is known to function in the exocytosis network, however, the molecular mechanism is unclear yet. Using UV/trimethylpsoralen mutagenesis, the sec-10 (one component of the exocyst complex) knockout mutant of C. elegans was obtained for the first time. The drug sensitive assays revealed clearly that the sec-10 gene affected the neural signal transmission, however, the electrophysiological assay showed the function of the ionotropic receptors in the neuromuscular junctions (NMJs) were unaltered compared with the wild type (WT). Thus it was assumed that the sec-10 gene might not influence the known ionotropic receptors in the NMJs, but some other pathways instead.

Abstract:Escherichia coli is the most common gram-negative organism causing neonatal meningitis. In order to characterize the role of the pathogenicity island gene ibeT of Escherichia coli K1 in the pathogenesis of neonatal meningitis, an ibeT gene isogenic in-frame deletion mutant strain was constructed. Intracellular survival assay in human brain microvascular endothelial cells (HBMECs) showed that the deletion of ibeT inhibited the growth of Escherichia coli K1 within HBMECs. Confocal microscopy analysis showed that much more ibeT mutant remained in lysosomes of HBMECs compared with wild type Escherichia coli K1 after bacterial invasion into HBMECs. Transmission electron micrographs further showed that ibeT mutant strain was failed to disrupt the Escherichia coli containing vacuole (ECV) and escape into the cytoplasm. Furthermore, cytoplasmic buffering capacities of ibeT mutant were lower than wild type Escherichia coli K1 at pH 6.5 and pH 6.0. These results suggested that the expression of ibeT in Escherichia coli K1 contributed to ECV membrane degradation and subsequent escape from lysosomes into the cytoplasm for replication after Escherichia coli K1 invasion into HBMECs.

Abstract:Tumor necrosis factor alpha(TNF-α) is a multi-functional cytokine that plays a significant role in many autoimmune diseases. The key biological functional domain of mouse TNF-α was determined by identifying the binding site of mouse TNF-α neutralizing antibody 9C6. Using yeast surface display technology, it was determined that 9C6 can recognize the linear amino acid fragment 29～40 of mature mouse TNF-α protein. Through mutagenesis experiments of this TNF-α region, the critical amino acids necessary for 9C6 binding were identified. Finally, wild-type mouse TNF-α and mutant variants that loses binding ability to 9C6 were expressed in Escherichia coli, purified, and used in a L929 cell killing assay. The assay results proved that the key amino acids for 9C6 binding were consistent with mouse TNF-α functional domain.

Abstract:Cytoplasmic male sterility (CMS) is the maternally inherited inability to produce functional pollen in a range of crop plants and has been widely used for the production of hybrid seed in plant breeding .To better under- stand the molecular mechanism on protein level and find the crucial proteins which related to fertillity, total anther proteins extracted from uninucleate and binucleate stage of (S)-1376A and (A)-1376B were separated by two- dimensional electrophoresis, the coomassie brilliant blue stained protein spots were analyzed using PDQuest software, among 610 reproducibly protein spots could be visualized on each 2D-gel within M 9.0～100.0 ku and pI 4～7. A total of 28 differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS), 5 of them were identified following NCBInr database queries, which are ubiqutin-protein ligase E2, putative glycine-rich protein, ascorbate peroxidase, putative cysteine proteinase inhibitor, ribulose bisphosphate carboxylase small chain clone 512. These proteins were involved in the process of energy metabolism, programmed cell death (PCD), regulating flower development.These results provide a clue for elucidating the mechanism of CMS.

Abstract:To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5₊CHO_- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1×10_-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22×10_-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

Abstract:Cancer cell metastasis is the major cause of lung adenocarcinoma (AdC) with high mortality and poor prognosis. To screen metastasis-associated biomarkers of lung AdC, laser capture microdissection (LCM) was used to purify the cancer cells from primary lung AdC with (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis. Then two-dimensional differential in-gel electrophoresis (2D-DIGE) was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC. The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry (MS). The partial differential proteins annexin A1, annexin A2, annexin A3, S100A9 and B23 were validated by Western blot. 2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established, and 20 differential proteins in the above two tissues were identified, of which 13 proteins were up-regulated and 7 proteins were down-regulated in LNM AdC compared to non-LNM AdC. Western blot results indicated that annexin A1, annexin A2, annexin A3 and S100A9 were significantly up-regulated in LNM AdC compared to non-LNM AdC; B23 was significantly down-regulated in LNM AdC compared to non-LNM AdC. Immunohistochemical analysis indicated S100A9 was up-regulated in LNM AdC compared with non-LNM AdC. It was the first time that metastasis-associated proteins were identified in primary adenocarcinoma by LCM coupled with 2D-DIGE and MS techniques. The findings will facilitate understanding of lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients’ outcome so as to improve the diagnose and treatment of lung AdC.

Abstract:Ten ATP binding cassette (ABC) transporters are confirmed to be associated with resistance against anticancer drugs. To investigate the relationship between these ten ABC transporters and the nasopharyngeal carcinoma cell line CNE2 resistant to cisplatin, cisplatin and cisplatin with 5-fluorouracil were used to induce the CNE2 cell to acquire the drug-resistance for 1 year. After these cells were cultured without drugs for 2 months, the MTT assay method was used to determine the dose-effect relationship of cisplatin and resistant index. Quantitative real time polymerase chain reaction was used to detect the mRNA expression of ten ABC transporters in CNE2 and the drug-resistant CNE2 cells, and the result was confirmed by immunocytochemical method. The results of MTT method showed that two cell lines resistant to cisplatin (named as CNE2/DDP) and cisplatin with 5-fluorouracil (named as CNE2/DDP+5Fu) were established, with resistant index 2.58 and 5.31, respectively. Of ten ABC transporters, only ABCC2 was found to be up-regulated both in CNE2/DDP and CNE2/DDP+5Fu cells, for increasing about 2.50 and 4.08 folds, respectively. The results of immunocytochemical method also confirmed that the expression of ABCC2 in CNE2/DDP and CNE2/DDP+5Fu cells were stronger that that in CNE2 cell. Furthermore, ABCC2 protein was found to be located at nuclear membrane of CNE2/DDP+5Fu cell but not at nuclear membrane of CNE2 cell. The results suggest that ABCC2 may play an important role in cisplatin- resistance of nasopharyngeal carcinoma cell line CNE2.

Abstract:In order to explore the function of EphA2 gene on the proliferation, apoptosis, motility and invasion of glioma cell line U251. The EphA2 mRNA levels of normal brain and two glioma cell lines U251 and U87 were detected by reverse transcription PCR (RT-PCR). Then, the function of EphA2 in glioma cell line U251 was analyzed by RNAi technology. The expression level of EphA2 gene was higher in U251 and U87 cells than that in normal brain. siRNA targeting EphA2 was designed and synthesized and then the siRNA was transfected into U251 cell with a high transfection efficiency of 70.31%. 50 nmol/L of siRNA could lead to over 70.0% reduction in EphA2 protein and mRNA level detected by Western blot and RT-PCR respectively. The cell proliferation was notably attenuated and the apoptosis was significantly increased after EhpA2 was downregulated. The migration and invasion of glioma cells were also attenuated when EhpA2 was knocked down by siRNA. EphA2 plays an important role in the malignance of glioma U251 cells and may be a novel molecular marker for the malignance of the glioma cell line U251. EphA2 could be a potential target in gene therapy for glioma.

Abstract:Glucose transporter 4 (GLUT4), a major contributor to glucose transport in skeletal muscle, is closely related to diabetic treatment. Exercise regulates apparently GLUT4 gene expression which produces many beneficial metabolic adaptations for diabetic patients. Study has shown that both AMPK (AMP-activated protein kinase) and CaMK (calcium-calmodulin dependent protein kinase) signaling pathways are involved in regulation of exercise-induced GLUT4 gene expression in skeletal muscles, but the relationship between these two signaling pathways in regulating the GLUT4 gene is unclear. The purpose of the following study was to investigate the relationship of these two pathways in Caffeine- and AICAR-stimulated skeletal muscle cells GLUT4 gene expression. Muscle contractile activity results in increases in both cytosolic Ca²⁺ and AMPK activity. To mimic this response, primary cultured rat skeletal muscle cells were treated with Caffeine to raise cytosolic Ca²⁺ and with AICAR to activate AMPK. The muscle cells were divided into different groups (Control, AICAR, Caffeine, AICAR/Caffeine, Caffeine+Compound C, AICAR/Caffeine+Compound C, AICAR+KN93, AICAR/Caffeine+KN93), which were used for experiments of stimulation by AICAR and Caffeine, inhibition by AMPK inhibitor, Compound C and CaMK inhibitor, KN93 respectively. The results showed that both AICAR and Caffeine induced about 2- and 3-fold increases respectively (P < 0.05, AICAR vs. Control; P < 0.05, Caffeine vs. Control) in GLUT4 mRNA in muscle cells, but the effect of GLUT4 mRNA induced by the combined stimulation of raising cytosolic Ca²⁺ and activating AMPK was not additive. Moreover, the Compound C, an AMPK inhibitor, decreased the Caffeine-induced increases in GLUT4 mRNA (P < 0.05, Caffeine+Compound C vs. Caffeine), and also attenuated an increase in GLUT4 mRNA induced by the combined stimulation of AICAR and Caffeine (P < 0.05, AICAR/Caffeine+Compound C vs. AICAR/Caffeine). Similarly, the Caffeine induced an increase in α1-AMPK phosphorylation (P < 0.05, Caffeine vs. Control) and furthermore the Compound C reduced apparently such an increase (P < 0.05, Caffeine+Compound C vs. Caffeine); however, KN93, a CaMK inhibitor, was completely able to inhibit the Caffeine-induced increase in GLUT4 mRNA, but failed to inhibit the AICAR-induced 2-fold increases in GLUT4 mRNA (P < 0.05, AICAR+KN93 vs. Control) and also failed to block an increase in GLUT4 mRNA induced by the combined stimulation of AICAR and Caffeine (P < 0.05, AICAR+Caffeine+KN93 vs. Control). These results demonstrate that the CaMK and AMPK signaling pathways are not complete independent, but are cooperative in the regulation of Caffeine-induced GLUT4 gene expression in cultured skeletal muscle cells. Collectively, based on our results and the other results this paper suggests that the GLUT4 gene expression in contracting skeletal muscle cells may be regulated through a CaMK-AMPK signaling pathway.

Abstract:Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. The timing of cognitive dysfunction and the relation of this cognitive impairment to calcium/calmodulin-dependent protein kinaseⅡ (CaMK-Ⅱ), plasticity related gene 3(PRG-3), cholecystokinin(CCK), zinc transporter 1 and 3 (ZnT-1 and ZnT-3) in hippocampus were studied. A seizure was induced by penicillin quaque die alterna in Sprague-Dawley rats from postnatal day 29 (P29). Rats were assigned into the recurrent seizure group (RS, seizures were induced in eleven consecutive days, n=39) and the control group (NS, n=21). At 1.5 h, 3 h, 6 h and 12 h after the last seizure, apoptosis and autophagy were detected by transmission electronmicro-scopy (TEM) or in situ end labeling (TUNNEL). Electro-corticogram was observed after the last seizure. During P51 to P56, P81 to P84 and P92 to P95, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. On P95, mossy fiber sprouting and gene expressions in hippocampus were determined subsequently by Timm staining and real time RT-PCR methods. The results are as follows: a. TEM revealed the formation of autophagosomes and apoptosis in hippocampus after kindling. In the hippocampal neurons of the controls, chromatin and cytoplasmic organoids showed eumorphism with nuclear membrane integrity; 1.5 h after the last penicilin-induced recurrent seizures, “C”-morphous double membrane structure of dilated endocytoplasmic reticulum could be observed; 3 h after the last seizures, autophagosome appeared with manifest dilated endocytoplasmic reticulum; 6～12 h after the last seizures, the hippocampal neurons showed apoptotic feature such as nuclear chromatinic pyknosis and verge-aggregation. TUNNEL staining also showed elevated number of apoptosis neurons in hippocampus of RS group(P < 0.05). b. Vertex sharp transient wave and sharp wave were soon detected just in a few minutes afer the last seizure in RS group by Electrocorticogram. c. Escape latency. The escape latencies from the water maze were significantly longer in rats of RS group than that of the control at d5 of the first test, at d1 of the second test, and at d2 of the third test. d. Strategy analysis. RIDIT analysis showed that the scores were much worse in rats of RS group than that of the control at d4 and d5 of the first test. Moreover, the scores were much worse in rats of RS group than that of the control at the whole three days of both the second and third maze tests. e. Memory test. As for the value of distance in origin platform quadrant to total distance, the RS group seemed worse than the NS group in three maze tests(P < 0.05). f. Aberrant mossy fiber sprouting was seen in the inner molecular layer of the granule cells and the stratum pyramidale of CA3 subfield in RS group. g. Real time RT-PCR analysis showed decreased expression of CaMKⅡα and ZnT-1 in hippocampus of RS group than that in control group. Moreover, the four genes (CaMKⅡα, PRG-3, CCK and ZnT-3) in NS group and the two genes (CaMKⅡα and ZnT-1) in RS group showed copolymerization characteristic by cluster analysis. In addition, correlation and regression analysis demonstrated positive linear correlation among CaMKⅡα, CCK, PRG-3 and ZnT-3 in NS group and between CaMKⅡα and ZnT-1 in RS group. It was concluded that recurrent developmental kindling induced by penicillin could cause not only autophagy and apoptosis in the earlier stage of brain damage, but also have long- term effects on cognitive function and hippocampal mossy fiber sprouting which may be associated with the down-regulated expression of CaMKⅡα and ZnT-1 in hippocampus.

Abstract:Cis9, trans11 and trans10, cis12-CLA are two major isomers of conjugated linoleic acid, which have stronger activities of anti-tumor. Based the previous studies, it was explored the pathway and mechanism of inducing apoptosis in human breast cancer cell line SKBR3 by c9, t11-CLA and t10, c12-CLA. It was confirmed that CLA could increase obviously the transcription and protein expression levels of PPARγ by RT-PCR and Western blot. The synchronism and correlation between PPARγ and apoptotic proteins Bax, Bcl-2, caspase3 changes were found with a dose- and time-dependent manner. PPARγ inhibitor GW9662 experimental result showed that there is cooperative relation between them. This is the first report that CLA induces apoptosis in SKBr3 cell by the signal pathway of PPARγ-Bcl-2-Caspase3. These observations indicated that CLA will be useful for clinic therapy of anti-tumor as a new regulator of PPARγ in the future.

Abstract:Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line, PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion] and RARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybrid technique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7 vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmid was proved by Western-blot. The transformed yeast AH109 was mated with yeast YI87 (containing leukemia cDNA library plasmids pACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After being reintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among which one containing JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence, GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration of NLS-RARα signaling to APL.

Abstract:A novel method for high-performance and high-resolution separation and detection of single-stranded DNA based on low-cost plastic microchips using electrophoresis apparatus equipped with dual wavelength confocal fluorescence detection have been developed. High-quality microchannels are fabricated on a thin poly (methyl methacrylate) (PMMA) plastic sheet (0.8 mm), ensuring superior limits of detection and good dissipation of Joule heat. A reproducible allelic profiling assay for the analysis of short tandem repeat (STR) was developed using replaceable denaturing linear polyacrylamide as the sieving matrix and cellulose derivates as adsorptive modifiers to suppress nonspecific adsorption of DNA samples to the PMMA surfaces. Under optimal conditions, all fragments of the STR allelic ladders of the D13S317 and CSF1PO loci as well as PCR-amplified samples from individuals can be separated unambiguously within 180 s. These results demonstrates the potential of plastic microchips for low-cost genetic analysis.

Abstract:The microchip fabricated by MEMS technology is successfully used to finish the electrophoresis process. A novel electrophoresis analysis protocol of the trace enzyme of lactate dehydrogenase(LDH) on microfluidic chip platform is developed with a Xe lamp-induced fluorescence detection system. Satisfactory separation of LDH was achieved in 75 mmol/L borate buffer containing 9.73 μmol/L calcium lactate as running buffer (pH 9.4) within 4 min. The detection of LDH limits (S/N=3) is 6×10^-3 U/L. The coefficients of variation of peak time and areas were 5.32% and 3.17%. The method is easily operated. To application microfluidic chips with the method, the sensitive of detectable enzyme will be much more improved．The method for trace enzymes has potential for clinical application.