Abstract:Apoptosis is an organized suicide program which is evolutionally conserved from yeast to mammals. Research on yeast apoptosis has made rapid progress, though it remained unrecognized until recent years. Initial observations show that yeast can be induced to undergo apoptosis and a number of conserved pro- and antiapoptotic proteins have been identified in Saccharomyces cerevisiae. Yeast has been validated as a model organism to investigate mechanisms of apoptosis. Recently, yeast has also been used as a model to study apoptosis-related disease, such as Huntington's disease and Parkinson's disease. The feasibility, the advantages and the perspectives of yeast model for apoptosis research are reviewed.

Abstract:Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of pluripotent cells, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into pluripotent cells. The present state of these reprogramming technologies will be reviewed and their relative success will be discussed.

Abstract:ATP-binding cassette transporter A1(ABCA1) is a kind of membrane intergrate protein and may have multiple and diverse functions. It mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoproteinA-Ⅰ(apoA-Ⅰ) and plays a significant role in high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-Ⅰ and HDL and prevalent atherosclerosis. ABCA1 expression is highly regulated and implies a variety of molecular actors. All of the nuclear receptors which involve in regulation of ABCA1 expression act via the DR4 element in the ABCA1 promoter. cAMP up-regulates ABCA1 expression by acting both at the transcriptional and translational level. Cytokines have been shown to exert pleiotropic and antinomic effects on ABCA1 transcription. In addition to these, some of enzymes and proteins such as protein kinase A, protein kinase CK2, cathepsin D are involved in the regulation of ABCA1 expression. The recent progress in the structure, function and regulation of ABCA1 transporter is reviewed.

Abstract:RIG-Ⅰ, the abbreviation of retinoic acid inducible gene-Ⅰ, can be induced to express in many type cells by various stimuli. Recently, it was identified as an intracellular regulator for RNA virus-induced antiviral response in innate immunity. Its discovery, expression induction, structure, research status of its function, homologous proteins and functional mechanism etc. were summarized, and its further research pulses are also prospected meanwhile.

Abstract:Chronic pain due to brachial plexus avulsion (BPA) is a kind of refractory neuropathic pain, yet there is a lack of knowledge regarding underlying brain activity. To further identify brain regions involved in chronic pain due to BPA, fluorine-18 fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography (PET) was used to observe brain glucose metabolic changes in those patients. Five patients with chronic pain due to left-BPA, whose pain reduced more than 75% after dorsal root entry zoneotomy (DREZotomy) were selected. The visual analog scale (VAS) , Hamilton depression scale, Hamilton anxiety scale and ¹⁸F-FDG PET of brain were recorded before and 14 days after DREZotomy respectively. Statistical parametric mapping 2 (SPM2) was applied for data analysis. Comparing with PET during pain before DREZotomy, PET after DREZotomy showed significant glucose metabolism decreases in bilateral caudate, orbitofrontal cortex (OFC) (BA11), contralateral subgenual cingulate (BA25) and ipsilateral dorsolateral prefrontal cortex (DLPFC) (BA46/47), and significant glucose metabolism increases in contralateral thalamus, pulvinar and ipsilateral parietal lobe (BA7). The results suggested that the brain areas involved in emotion, attention and internal modulation of pain play an important role in the modulation of chronic pain due to BPA.

Abstract:Formaldehyde is directly toxic to cells and its intermediate metabolite formic acid leads to acidosis in microenvironment in vivo. According to recent literatures, endogenous formaldehyde production is related to several metabolic pathways such as amine oxidation (catalyzed by semicarbazide-sensitive amine oxidase, SSAO), methylation and demethylation. Under oxidation stress and energy metabolic imbalance, formaldehyde markedly increases in human circulation. Here, the authors found that formaldehyde is released from the reaction of malondialdehyde with a protein (BSA) in which protein side chains such as amino groups are chemically modified. Moreover, formaldehyde is also produced from the sphingomyelin solution in the presence of hydrogen peroxide as the myelin peroxidation occurs. Formaldehyde at low concentration induces neuronal Tau aggregation, resulting in formation of globular like aggregates which are toxic to SH-SY5Y cells, HEK-293 cells and hippocampus neurons in the primary culture. According to Chen et al. (2006), endogenous aldehydes are related to beta-amyloid misfolding, oligomerization and fibrillogenesis. Furthermore, formaldehyde is able to react with some neurotransmitters and thus impairs their structures and functions. Under physiological conditions, the human blood formaldehyde is dynamically kept approximately (0.087±0.004) mmol/L. Notably, this concentration is close to the half-lethal dose of formaldehyde (0.10～0.12 mmol/L) to neural cells in the in vitro cell culture such as SH-SY5Y cells. Furthermore, cell growth can be partially affected and inhibited in the presence of formaldehyde at (0.087± 0.004) mmol/L during the in vitro culture. This suggests that human body needs a strong degradation system to remove endogenous formaldehyde. As shown in clinical trials, the formaldehyde level in urine of Alzheimer's patients was markedly higher than the control subjects. The urine formaldehyde level was shown to be related to the cognitive impairment. Therefore, the level of blood (brain) formaldehyde is supposed to be changeable and increased under aging, leading to a higher risk chance to impair human brain, especially under stressing. The formaldehyde chronic damage to neural cells (grey mater) and neural fibers (white mater) may be one of the most important pathological mechanisms for sporadic neurodegeneration for instance Alzheimer's disease, because hypofunction in scavengering endogenous formaldehyde occurs as aging.

Abstract:Mass spectrometry is being widely applied to identify and quantify proteins in complex mixtures. Quantification of small molecules by integration of LC-MS extracted ion chromatogram (XIC) peaks has a long history in analytical chemistry. Similar quantification techniques applied to proteolytic protein digests have also been previously described. A comprehensive approach for label-free quantification using yeast proteome as a model have been developed. Based on spectra counts of peptides, the relative protein quantification from LC-MS/MS experiments of proteolytic protein digests was performed. Unlabeled protein samples were digested with trypsin and separated by one-dimensional nano-flow HPLC (RPLC), and mass spectra were obtained by using the survey mode of LTQ mass spectrometer with dynamic exclusion. The correlating relationship between concentrate of protein and spectra counts was confirmed. Two algorithms to normalize spectra counts ratio from different samples were compared, and the results suggested that algorithm based on average spectra count (ASC) was eximious. The method was used to biomarkers discovery in HepG2 and HepG2-HBx cell lines. The identified proteins were analyzed and classified by cluster software (Version 3.0). Finally, 107 overlap proteins were identified, among them, 9 proteins were identified up-regulated (Ratio > 1.75) and 6 proteins down-regulated (Ratio < 0.5). Further research indicated that these proteins were related with liver cancer. Altogether, the results indicated that the strategy was operable and convenient with high sensitivity and wild dynamic range, and will be significant to deliver biomarker discovery either in theory or in clinic.

Abstract:To screen for methylation silenced genes in nasopharyngeal carcinoma cell line 5-8F, two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with demethylating agent 5-aza-2-dC, PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins between the treated and untreated 5-8F cells. Then RT-PCR and Western blotting were performed to examine the expression levels of nm23-H1 mRNA and protein, one of the differential expression proteins, in the treated and untreated 5-8F cells, respectively. Methylation-specific PCR (MS-PCR) was performed to detect the methylated level of nm23-H1 gene in the treated and untreated 5-8F cells. 2-DE patterns of the treated and untreated 5-8F cells with 5-aza-2-dC were established, and a total of forty-nine differential protein spots were found in treated and untreated 5-8F cells. Thirty-three non-redundant differential proteins were identified by MS, 15 proteins of which were up-regulated after 5-aza-2-dC treatment. The results of Western blotting, RT-PCR and MS-PCR showed that nm23-H1 is a methylation silenced gene in 5-8F cell line. Encoding genes of 15 up-regulated proteins after 5-aza-2-dC treatment may be methylation silenced genes in NPC cell line 5-8F. The data will be helpful to screen for methylation silenced genes in nasopharyngeal carcinoma.

Abstract:With the success of the human genomic project, the amount of experimental data is increasing rapidly. In the post-genomic era, the focus is now shifting to the so called "from the sequence to the function", i.e., in addition to completing genome sequences, it is possible to learn about gene expression patterns and protein interactions on the genomic scale. Description and analysis of complex Genetic Regulatory Networks (GRN) is a critical problem for biologists to understand genetic regulatory mechanism. Most of existing methods ignore the synergistic effects observed widely in biologic systems. Thus there should be some errors between the predictions of the model and the actual biologic behaviors. To address this problem, a new quantitative analysis approach for genetic regulatory networks was proposed in the context of Hybrid Functional Petri Net (HFPN). Firstly, basic theory of GRN and HFPN was presented briefly. Petri-net-based models have been widely adopted for studying biological systems since Petri net has graphical modeling representation and strict mathematic background. In GRN, the combined effect of transcription factors to induce or repress gene transcription is usually different from the simple sum of their individual effects. This is so called Synergistic effect. Two kinds of new elements, logic places and logic transitions were introduced to describe the logic rules in GRN and the synergy between transcription factors. It was decided to extend existing tools instead of writing new software in order to have more time for the experimental part of this project. A Petri Net Workbench was developed based on the Open Source project the Petri Net Kernel (PNK) and the Systems Biology Workbench (SBW) during this project. The Petri Net Workbench was extended by the following features: the extended definition of Hybrid Functional Petri Net, the Ordinary Differential Equations (ODEs) solver, Systems Biology Markup Language (SBML) support and time course simulation. Finally, a Petri net model for the genetic regulatory network of the sea urchin endo16 gene was developed base on the GRN model published in literature and a list of quantitative outputs for different mutations was predicted. The analysis result corresponded to the experimental data published in literature properly and demonstrated the correctness of the Petri net model. It demonstrated how Hybrid Functional Petri net analysis techniques can be applied to Genetic Regulatory Networks. The new concepts of logic places and logic transitions had been proven to be useful for describe the logic rules in GRN and the synergy between transcription factors.

Abstract:Using LPS mediated-endotoxemia BalB/C mice, the role of heat shock factor 1 (HSF1) in heat shock response (HSR) was observed. HSR was performed with 42℃ for 15 min, and recovery for 24 h at room temperature. Endotoxemia model in mouse was achieved by intra-peritoneal injection of LPS at 14 or 15 mg/kg. Lung injury and expression of inflammatory mediators were evaluated with myeloperoxidase (MPO) and maleic dialdehyde (MDA) in heart and lung, RT-PCR, hemotoxylin-eosin (HE) staining and mortality. The data showed that the survival rate was higher in HSR+LPS (HSF1+/+) group (7/15) than that in LPS (HSF1+/+) group (0/15), LPS (HSF1－/－) group (0/14) or HSR+LPS (HSF1－/－) group (0/14) within 72 h after injection of LPS at 15 mg/kg. Similarly, the survival rate was also higher in LPS (HSF1+/+) group (5/15) than that in LPS (HSF1－/－) group (0/13) or HSR+LPS (HSF1－/－) group (0/13) within 72 h after injection of LPS at 14 mg/kg. HSR significantly suppressed production of MPO and MDA induced by LPS in lung and heart in HSF1+/+ mice, but had no such effects in HSF1－/－ mice after 12 h treatment with 14 mg/kg LPS. The inflammatory mediators, including SOCS3, MCSF, GCSF, IL-1β, IL-6, CCL-2 and IL-15 were up-regulated both in HSF1+/+ and HSF1－/－ mice after12 h treatment with LPS at 14 mg/kg, and HSR repressed LPS-induced up-regulation of SOCS3, MCSF, GCSF, IL-15, IL-6 and of CCL-2 in HSF1+/+ mice, but not in HSF1－/－ mice. HE staining indicated that LPS at 14 mg/kg could mediate significant morphological changes, including necrosis, intravascular coagulation and leukocytes aggregation, and adherence in lung, liver and kidney in HSF1+/+ and HSF1－/－ mice. The morphological changes in these organs were attenuated with HSR in HSF1+/+ mice, but exacerbated in HSF1－/－ mice. Those results suggested that HSF1 knock out could significantly block the protection of HSR against LPS mediated-endotoxemia in BalB/C mice.

Abstract:As a candidate for progenitor of endocrine cells, pancreatic duct epithelium displays more and more importance of differentiation potential to insulin-producing cells. Some cell markers such as pdx-1, nkx6.1 expressed in pancreatic islet development have also been found in duct. The maf family, which is a subgroup of the basic leucine zipper(bZip) transcription factors with homology to the v-Maf oncoprotein, the founding member of the Maf family, which was originally identified in the genome of the AS42 chicken musculoaponeurotic sarcoma retrovirus in 1989, has been involved in pancreatic research recently. Members of Maf family, mafa, mafb, c-maf, which have been reported to be regulators of tissue-specific gene expression and cell-differentiation in a wide variety of tissues, are all expressed in islet and regulate insulin expression as well as islet development. Whether these genes are expressed in duct remains unclear and demands further investigation. Islet, duct and exocrine tissues were acquired by laser-capture microdissection from mouse pancreatic section, followed by real-time PCR to quantitate the target genes expression. Mafa, mafb, c-maf were all detective in duct and islet but none was in exocrine tissue. In addition, c-maf mRNA level in duct was higher than islet(0.300±0.018 vs 0.796±0.173, P < 0.05), however, there was no difference in mafa and mafb mRNA level between islet and duct. The expression of mafa, mafb, c-maf in pancreatic duct epithelium, gives more support to not only the roles of the three genes in endocrine development, but also the potential of converting duct epithelium to endocrine cells. Moreover, the higher expression of c-maf in duct than islet suggests that down-regulation of c-maf might help to differentiation and mature of endocrine cells.

Abstract:During the process of pathogens penetrating the plant cell, pathogens often secret some chemicals into plant cells, at the same time, they also produce mechanical signal by physical pressure on the plant cell. Here the pressure is used as the stress signal, to study its effect on phytoalexin accumulation and the induction of plant resistance in cucumber seedling. It is found that stress can induce the resistance in cucumber seedling significantly. When breaking the plant cell wall and plasma membrane adhesion by RGD peptides, the resistance induction is almost eliminated. Results from TLC and HPLC showed that stress stimulation could increase phytoalexin accumulation in cucumber seedling. This suggests that the accumulation of phytoalexin is one possible reason of the increased resistance after stress stimulation. When the adhesion between plant cell wall and plasma membrane was block by RGD, there is only small amount of phytoalexin accumulation compared with the control, suggesting that the stress induced phytoalexin accumulation and resistance is relying on the adhesion of plant cell wall and plasma membrane.

Abstract:Kidney samples were obtained from db/db mice and their db/m littermates at the age of 4, 8, 12, 16, 20 and 24 weeks. The expression of ANGPTL2 was assessed by RT-PCR and immunohistochemistry, and then correlated with biochemical and histological indices of kidney injury. No significant difference of ANGPTL2 expression was found in the kidney of db/db mice and the control db/m mice at the age of 4 weeks, a time when all biochemical and histological indices indicated that the db/db mice were in pre-diabetic condition. However, with the development of obesity, hyperglycemia and proteinuria, the expression of ANGPTL2 in the kidney of db/db mice increased, which indicated that the increased expression of ANGPTL2 associated with diabetic nephropathy. ANGPTL2 protein was found distributed mainly in the glomerulus. It is along the capillary loop, located just outside the basement membrane, and in the same location as podocytes, which suggested that podocytes are the main origin of ANGPTL2 in the kidney. Besides, increased expression of ANGPTL2 was found in older db/m mice though it has not reached to a significant level, which may suggest that ANGPTL2 might have some thing to do with the kidney injury caused by ageing.

Abstract:The bacteriophage T7 RNAP gene was amplified via PCR from λ-lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

Abstract:Previous work from this laboratory has cloned a novel gene NOR1 and showed its extensive expression in normal tissues and down-regulation in carcinomas. To further investigate its downstream target genes and better understand its function, NOR1 was over-expressed in HepG2 hepatoma cells and global changes in gene expressions from a stable line were identified by cDNA microarrays. The results discovered 59 genes up-regulated in these cells compared with the original cells, including Grb2, HBP17, TNFRSF11B genes that have been implicated in tumorigenesis and cancer development. In addition, 103 down-regulated genes were also identified, including genes encoding Bik, MAP2K6 and ZFP95 proteins. The expression patterns of certain genes identified by microarrays were validated by quantitative real-time PCR and the results showed that expression difference were statistically significant (P < 0.05). These data suggest that NOR1 may influence the biology and cancerous behaviors of HepG2 cells by regulating expression of a set of genes involved in signal transduction, cell cycle regulation, transcription and translation controls.

Abstract:Previous research revealed that distortion is detected in transient voltage signal recorded with traditional patch clamp amplifier under current clamp mode, which is essentially resulted by electronic design of the headstage of the patch clamp. A new kind of headstage is designed to modify the defect, the circuit of which not only measures the transient potentials as the classical voltage follower does but also is quite suitable for the standard voltage-clamp mode. Furthermore, the technique of voltage-clamp-controlled current-clamp is applied for modifying the conventional patch-clamp amplifier, the variable low-pass filter is added into the circuit to reduce the response speed of voltage-clamp module, thus the transient potentials changes can be measured while membrane potential is kept at a constant value. Bridge balance circuitry is designed to eliminate the voltage drop while the variable current injected into the electrode. And fast capacitance compensation stage of conventional PCA is modified to neutralize the capacitance and accelerate system response speed for current-clamp mode. The experimental results on cell model demonstrate that modified PCA meets the requirement of monitoring transient potential changes in electrophysiology research.

Abstract:A dual-label time-resolved fluoroimmunoassay was established for simultaneously detecting pepsinogen Ⅰ (PGⅠ) and pepsinogen Ⅱ (PG Ⅱ) in human serum. Two capture monoclonal antibodies, 8003^# of PGⅠ and 8101^# of PGⅡ, were co-coated in 96 microtitration wells. The counterpart tracer monoclonal antibodies, 8016^# of PGⅠ and 8102^# of PGⅡ, were labeled with Eu³⁺ and sm³⁺-chelates, respectively. The samples were assayed by one-step sandwich protocol with the time-resolved fluorometry. The measurement ranges of PGⅠ were 0.2～300.0 μg/L with the within-run and between-run precision was 5.2% and 8.1%, and that of PGⅡ were 0.05～55.0 μg/L with the within-run and between-run precision was 7.1% and 11.7%, respectively. The average recovery rates of PGⅠ and PGⅡ were 96.9% and 103.7%, respectively. The results obtained by the dual-label assay agreed well with those by enzyme-linked immunosorbent assays of PGⅠ and PGⅡ, whose correlation ratio were 0.9426 of PGⅠ and 0.9396 of PGⅡ, respectively. The means of 300 healthy volunteers were (157.3 ±51.0) μg/L for serum PGⅠ,(10.6 ± 5.9) μg/L for serum PGⅡ, and (14.8 ± 4.3) for the PGⅠ/PGⅡ ratio. The normal ranges of serum PGⅠ levels for healthy volunteers were 55.3～259.3 μg/L, those of serum PGⅡ levels were less than 23 μg/L, the PGⅠ/PGⅡ ratio was more than 6. The proposed dual-label TRFIA for simultaneous detection of PGⅠ and PGⅡ is a simple, sensitive, and rapid method. It could provide serology high-screening of the samples for gastric diseases and would allow investigations into the possible diagnostic value of analysis in various clinical condition.