Abstract:Atomic nuclei in a strong magnetic field rotate at a frequency that is dependent on the strength of the magnetic field. Their energy can be increased if they absorb radio waves with the same frequency (resonance). When the atomic nuclei return to their previous energy level, radio waves are emitted, called nuclei magnetic resonance (NMR). Based on this principle, knowing that the energy released attenuates at different rate in different materials, images of internal structures of an object can be acquired using magnetic resonance imaging (MRI) by applying a gradient and detecting radio wave signals which reveal the positions and kinds of certain nuclei constituting the object. The attempts of utilizing this technique on human by P.Lauterbur and P.Mansfield have led to a revolutionary tool for medical diagnose. Extremely rapid imaging could be achieved by very strong and fast gradients, which helped realizing MRI in medical imaging and research, and promoted the development of medicine, neurophysiology as well as cognitive neuroscience remarkably.

Abstract:The Royal Swedish Academy of Sciences announced that The Nobel Prize in Chemistry for 2003 is shared between two USA scientists in honor of their discoveries concerning channels in cell membranes. Agre discovered water channels of the cell membrane, and clarified important properties such as the selectivity for water molecules of the water channels. MacKinnon clarified the 3D structure of the K⁺ channels with high resolution, and elucidated its functional mechanisms such as ion selectivity in detail. The two scientists utilized their high sensitivity for the top scientific research and methodology. They started their work from the basic research of biochemistry, and made excellent contribution to the post genome study, which is the top area in the field of life sciences.

Abstract:Peptide antibiotics from plants are a type of small-size peptides which at least display antimicrobial property in vitro. The expression of some of them can be induced by many factors such as bacteria, fungi and other physical and chemical stimuli, others are constitutively expressed. According to the nucleotide sequences and their secondary-dimensional structures, nine families of peptide antibiotics have been identified in plants. These include thionins, defensins, so-called lipid transfer proteins, hevein- and knottin-like peptides, four cysteine-type, and the recently reported shepherdins, snakins and cyclotides. Recent advances concerning the classification, mode of action, biological properties and plant genetic engineering with some of peptides antibiotics are summarized in an attempt to promote the research and development in this field in China.

Abstract:Neuroglobin (NGB) is a newly discovered member of the hemoglobin family that reversibly binds O₂ and is expressed predominately in vertebrate brain. NGB is a monomer composed of 151 amino acids, with high oxygen affinity. It is of ancient evolutionary origin. Hithertos, it has been proved to associate with oxygen storage, transport and neuron protection. NGB gene expression is mediated by at least 2 signal transduction pathways. The discovery of NGB provides a new direction for treating patients with strokes and other hypoxia-related diseases.

Abstract:The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the unique amphipathic α-helical peptide identified in humans up to date. LL-37 is not only a major protein of blood cells, but is also present in epithelial cells. This peptide has a broad spectrum of antimicrobial activity, which kills bacteria by disrupting membrane according to the “carpet like” mechanism. LL-37 possesses the ability to bind lipopolysaccharide(LPS) and neutralizes its biological activity via its binding activities for LPS and CD14. In addition, LL-37 acts as a chemoattractant to recruit immune cells to site of infection by binding to formyl peptide receptor-like 1 FPRL1. Taken together, LL-37 is a multifunctional modulator of innate immune responses and might have the therapeutic potential for the treatment of bacterial infection and immunocompromise.

Abstract:The regional high distribution of D-serine in mammalian central nervous system is consistent with that of N-methyl-D-aspartate receptor. D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes D-amino acids. D-serine as a glia-derived transmitter not only questions the basic ideas about “neurotransmitter” but also offers a novel way to treat some brain disorders as both over-stimulation and down regulation of NMDA receptors has been implicated in a large number of acute and chronic neurodiseases.

Abstract:Ingestion by phagocytes is the fate of most cells that undergo apoptosis. During apoptosis, there are many changes on the surface of apoptotic cells, including the exposure of phosphatidylserine, the alteration of membrane carbohydrates and the redistribution or clustering of glycoproteins, which are leading to recognition and uptake by phagocytes. Many engulfment receptors have been implicated and appear to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. The process of uptake may vary with the apoptotic and engulfing cell types. At least seven engulfment genes in C.elegans have mammalian equivalents, and represent elements of signaling pathways involved in uptake, which have been proposed to define two parallel and partially redundant pathways. The mutation of engulfment genes can change the process of apoptosis. The defections of phagocytosis can affect the body's normal immune response.

Abstract:Sex-determining genes have been identified in flies, worms and mammals but not, until recently, in nonmammalian vertebrates. The characterization of DMY gene in medaka is introduced and the significance of this affair is discussed. Studies about the existence of this gene in other fish species are also been introduced. Finally, direction for future research is suggested.

Abstract:Hepatic stem cells have an ability of self-renewal and can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells under various conditions. There are many factors such as microenvironment, cytokines and extracellular matrix that control hepatic stem cell differentiation. Differentiation of hepatic stem cells into hepatocytes is regulated by transcription factors and signal pathways, and hepatocellular carcinoma may result from the abnormal differentiation of hepatic stem cells. The plasticity, multi-potential differentiation, the mechanisms governing hepatic stem cell differentiation, and the relationship between hepatocellular carcinoma and the differentiation of hepatic stem cells are discussed.

Abstract:Human internal thoracic artery cells (HITASY) were used to explore the vascular smooth muscle cells (SMCs) converted from a synthetic into a contractile phenotype characterized by serum withdrawal, which may contribute to neointimal formation and restenosis after vascular injury.Confluent monolayer of HITASY cells cultured in M199 serum-free medium exhibited morphological and functional characteristics consistent with a differentiated phenotype. Smooth muscle cell contractile phenotype was determined by observing the expression of smooth muscle myosin heavy chain (SMMHC, a marker of fully differentiated smooth muscle) and smooth muscle alpha-actin (a marker for all smooth muscle, including immature smooth muscle).Serum withdrawal induced a prominent decrease in extracellular matrix protein synthesis resulting in a 58% lower cell number. Nonsignificant proliferation HITASY cells treated with platelet derived growth factor (PDGF) was detected. Western blot analysis revealed a reversible upregulation of smooth muscle α-actin, calponin, caldesmon and SMMHC. Further, RT-PCR also indicated the expression of smooth muscle alpha 22 gene in HITASY cells after serum withdrawal. Vasoactive agonist stimulated robust calcium oscillations that coupled cell contraction in HITASY cell area or length on average.Differential display PCR was used to screen differential expression genes in HITASY cells, and the data showed that HITASY cells have the potential to express cellular repressor of E1A-stimulated genes, only after cultured in medium serum-free. Following readdition 10% serum, SMCs underwent a reversible dedifferentiation, followed by proliferation. These findings support the pivotal role of SMCs phenotype modulation in vitro.

Abstract:An outwardly rectifying chloride channel (ORCC) was characterized in acutely dissociated hippocampal CA1 pyramidal neurons of adult rat by using inside-out configuration of patch clamp. The channels were usually activated by a long-lasting and depolarizing voltage step. Average single channel conductance was (16.58±1.54) pS for membrane potential between －60 mV and 0 mV, and (40.92±3.17) pS between 0 mV and +60 mV in symmetrical 150 mmol/L NaCl solution (n=10). The channel open probability was voltage dependent (V_m=－60 mV，P_o=0.44±0.12；V_m=+60 mV，P_o=0.86±0.06, n=10). The reversal potential of the channel was (－4.17±1.84) mV in symmetrical Cl^－ concentration (150 mmol/L). When partially substituting Na-Gluconate for NaCl, the reversal potential shifted to (－34.23±4.86) mV (［Cl^－］_i/［Cl^－］_o=(30 mmol/L)/(150 mmol/L)). This shift indicates a high Cl^－ selectivity of the channel. Chloride channel blockers DIDS and SITS showed a reversible inhibiting effect on the channel activity. These results provide a report of an outwardly rectifying chloride channel (ORCC) in hippocampal CA1 pyramidal neurons of adult rat.

Abstract:The homo-dimer, homo-trimer, homo-tetramer and homo-hexamer of protein were classified using both of support vector machine and Bayes covariant discriminant methods. It was found that the total accuracies of “one-versus-rest” and “all-versus-all” are 77.36% and 93.43% respectively using support vector machine in jackknife test, which are 26.72 and 42.79 percentile higher respectively than that of Bayes covariant discriminant method in the same test. These results show that the support vector machine is a specially effective method for classifying the higher protein homo-oligomers from protein primary sequences. Using “all-versus-all” policy is better than “one-versus-rest” policy for classifying homo-oligomers based on the same machine learning method (such as support vector machine). And it was also indicated that the primary sequences of homo-oligomeric proteins contain quaternary information.

Abstract:Calpain is a calcium-activated protease and there are two ubiquitously distributed mammalian calpains, namely calpain 1 (μ-calpain and CAPN1) and calpain 2 (m-calpain and CAPN2). Calpains regulate the function of many proteins by limited proteolysis. To determine the nature of different subtypes of calpain on degradation of microtubule-associated protein tau, the rat brain cortex extracts were incubated with 0.2 mmol/L, 1 mmol/L, 3 mmol/L and 5 mmol/L of CaCl₂ for 15 min at 37℃. The findings were that Ca²⁺ treatment at concentration 1～5 mmol/L led to significant proteolysis of tau protein and this degradation was blocked by calpain inhibitor, calpeptin. In addition, when the extracts containing 1 mmol/L CaCl₂ were treated with μ-calpain inhibitor (0.05 μmol/L of calpastatin) or m-calpain inhibitor (100 μmol/L calpain inhibitor Ⅳ) or both, the Ca²⁺-induced degradation of tau protein was decreased to 8.6%，92.5% and 97.8%, respectively. These data suggest that both μ-calpain and m-calpain in brain cortex extracts are activated by Ca²⁺ and both of them degrade tau protein, although, m-calpain plays a more important role in proteolysis of tau.

Abstract:A full-length cDNA amplification technique based on the cap-finder method was used to obtain amplified amount of double strand cDNAs of the endometrial mRNAs at the implantation site from pregnant rhesus monkey. Then a subtractive library of the implantation site was successfully constructed by suppression subtractive hybridization (SSH). Of the randomly selected clones from the library，27% were proved by dot blot analysis to be differentially expressed ones at the implantation site. The data demonstrated that a subtractive library with high quality could be constructed from small amount of rare specimens by the combination of suppression subtractive hybridization（SSH）with full-length cDNA amplification technique based on the cap-finder method.

Abstract:A novel mouse heart-specific gene, Srd5α2l2, was cloned from mouse embryo heart by EST database searching and RT-PCR. The cDNA of Srd5α2l2 was 2 348 bp and composed of 12 extrons within over 77 kb in mouse genome. This gene was mapped to mouse chromosome 5 by BLAST search to mouse genome. Analysis of the 3′ untranslation region of the cDNA showed the presence of ATTTA sequence, which was believed to act as degradation signal of mRNA. The longest ORF spanned all of the 12 extrons and encoded a putative protein of 361 amino acids. A conserved steroid 5 alpha-reductase domain (3-oxo-5-alpha-steroid 4-dehydrogenase，STEROID_DH) was present at the C-terminus of the deduced protein. Homologous alignment found high identity of nucleotide sequence, deduced amino acid sequence and genomic structure between Srd5α2l2 and human cDNA of DKFZp313D0829（AL833108）. It may be concluded that AL833108 was the human orthologue of Srd5α2l2. BLAST search showed that cDNA of Srd5α2l2 was supported by 41 ESTs from EST database, 25 out of the 41 ESTs were from mouse heart. RT-PCR performed on a panel of different mouse tissues demonstrated that Srd5α2l2 strongly expressed in mouse heart but not or much more weakly expressed in other tissues. These results suggested that Srd5α2l2 is a novel heart-specific member of steroid 5 alpha-reductase family. As far as it is known that no heart-specific member of this family was reported before.

Abstract:Preparation of oligonucleotide microarray for gene expression detection of tumor-associated gene and its primary application in study on molecular mechanism of antisense oligonucleotide “cantide” were carried out. An oligonucleotide microarray consisting of about 450 kinds of tumor-associated genes was constructed and the standards of quality controls of it were founded at the same time. Tumor cells were treated with “cantide” mixed with lipofectin and total RNA was extracted. Through reverse-transcription, cDNA was synthesized and labeled with Cy5 or Cy3. After hybridized with the prepared microarray, signal intensities were detected and expression profiles were analyzed by software. The results indicate that the oligonucleotide microarray prepared has high specificity,good sensitivity and stablity etc. Compared to cells treated with lipofectin alone, the mRNA expression levels of seven genes appeared to show significant change. The mRNA expression of MDNCF , DHS genes were downregulated,while the mRNA expression of MUC2,MPP11,LAT,HRIF-B,JNK3A1 genes were upregulated after HepG2 cells treated with “cantide” for 15 h. The data should provide some candidate genes that may be involved in antitumor effect of “cantide”, and it is helpful to make a further investigation on the molecule mechanism. It can be concluded that the prepared oligonucleotide microarray can be used to investigate the gene expression profile of tumor-associated genes and provide techonical paltform for clinical diagnosis and basic research.

Abstract:Abnormal AKR1C2 expression has been observed in many malignant human tumors, but its relationship with hepatocellular carcinoma(HCC) is not well understood so far. In order to evaluate hepatocarcinogenic effect of AKR1C2 gene and the significance of its abnormal expression in hepatocellular carcinoma, AKR1C2 gene is analysed by preparing rabbit anti-human AKR1C2 polyclonal antibody, constructing of AKR1C2 frameshift mutant and exploring RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot, cDNA expression microarray, co-immunoprecipitation and the tumorigenicity assay in vivo and in vitro etc. AKR1C2 gene expression and its effects was analyzed, including 68 pairs of HCC specimens and its adjacent para-cancerous tissues, 8 cases of normal liver tissues and QGY7703 cell line. Results showed AKR1C2 expression was up-regulated among these patients, when compared with those of para-cancerous and normal liver tissues. Over- expression of AKR1C2 is also found to be correlated with high metastasis potentiality of HCC. AKR1C2 overexpression stimulates DNA synthesis, apoptosis, growth in soft agar and promote tumor formation and lead to expression differences of tumor genes. AKR1C2 mediated the NF-κB-dependent resistance of QGY7703 cells to anti-fas killing. Intracellar binding of AKR1C2 and Cdk4 was found. Abnormal expression of AKR1C2 gene may contribute to the occurrence, advancement and invasiveness of HCC from Qidong, China, a liver cancer high risk area.

Abstract:In the third step of the α-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde, which is regulated by 2-hydroxypanthyl-CoA lyase HPCL2. Using shot-gun sequencing technique, the genome sequence of the gene (HPCL2) code for the lyase was obtained. The gene is 40 829 bp in genome size including 17 exons and 16 introns, with an average 116 bp in exon size and 2 429 bp in intron size. The different transcriptional isoforms were also investigated by using method of alternative splicing in silica. In order to investigate the level of alternative splicing and to search for novel splice variants, total 213 ESTs derived from 29 different tissues were collected and aligned against the genome sequence of the gene. 17 ESTs were found having different splicing type that could be classified into three types of alternative splicing. 14 of the ESTs were detected to have exon skipping, 2 intron not spliced and 1 splice site shift. The data suggested that exon skipping was possibly one of the major mechanisms of transcription regulation of HPCL2 gene.

Abstract:In order to explore the role of Smac/DIABLO in H₂O₂-induced apoptosis of C₂C₁₂ myogenic cells, Hoechst 33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe DNA fragmentation. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blotting. Activities of Caspase-3, Caspase-9 were assayed with Caspase Colorimetric Assay Kit and Western blotting. Full length Smac/DIABLO gene was transiently transfected in C₂C₁₂ myogenic cells by lipofectamine and then protein levels of Smac/DIABLO were analysed by Western blotting. The results showed that: (1)H₂O₂ (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondrial to cytoplasm 1 h after treatment, activation of Caspase-3, Caspase-9 4 h after treatment and specific morphological changes of apoptosis 24 h after treatment; (2)Smac/DIABLO overexpression significantly enhanced H₂O₂ induced apoptosis in C₂C₁₂ myogenic cells as shown by specific DNA ladder pattern in agarose gel electrophoresis, increase of percentage of apoptotic nuclei and marked activation of Caspase-3, Caspase-9. These data suggested that H₂O₂ could result in apoptosis of C₂C₁₂ myogenic cells，and that release of Smac/DIABLO from mitochondrial to cytoplasm and the subsequent activation of Caspase-9 and Caspase-3 played important roles in H₂O₂-induced apoptosis in C₂C₁₂ myogenic cells.

Abstract:BLAST analysis suggested that a cDNA fragment (GenBank accession number BG222624) derived from human nasopharynx might represent a novel human gene. Applying the bioinformatics and experimental technique, a novel human gene have been cloned from the fetal brain cDNA library. Since this fragment contained a complete open reading frame(ORF) of 1 254 bp with a stop codon in its upstream and poly(A) signal in its downstream, it could be concluded that it is a full-length gene (GenBank accession number AY174896), which was named as adult retina hypothetical protein(ARHP). The full-length cDNA of ARHP gene is 1 672 bp, coding for a 417 amino acids polypeptide with a predicted molecular mass of 46.58 ku and isoelectric point of 4.20. The deduced amino acid showed 70% homology with a M.musculus protein BAB32214. Bioinformatics analysis suggested that the protein may be a nucleus protein regulating gene transcription. The new gene is comprised of four exons, with three intervening introns and it is localized to chromosome 5q35. It have been found that there are two CpG islands in 5′ UTR of this gene.

Abstract:To prepare and evaluate the clinical application of protein-chip for different HCV antibody detection, 905 serum samples were collected from three different hospitals. The samples were detected by ELISA method and protein-chip method respectively. Inconsistent samples were proved by imported HCV RIBA diagnostic kit. The results showed that (1)In all 905 serum samples, 294 positive samples and 611 negative samples were detected by ELISA method. Among the 294 positive samples, 292 positive samples and 2 negative samples were detected by chimeric antigen on the protein-chip and 288 positive samples, 2 negative samples, 4 indefinite samples were detected by the 4 segments of HCV antigens on the protein-chip. Among the 611 negative samples, 611 samples show negative result detected by chimeric antigen on the protein-chip and the 4 segments of HCV antigens on the protein-chip.The coordinate ratio of positive results are 99.3% and 98.9% respectively compared with ELISA method. The coordinate ratio of negative result are the same 100%. The 6 samples that were positive detemined by ELISA method and non-positive by protein-chip method were detected with RIBA reagent. All the 6 samples show non-positive result. (2)290 samples were detected with RIBA reagent. Among the 290 samples, 104 samples showed positive result, 66 samples showed positive result to single antigen segment, and 120 samples showed negative result. The 290 samples were also detected by protein-chip method. Among the 290 samples, 103 samples showed positive result, 61 samples showed positive result to single antigen segment, and 126 samples showed negative result. The result shows high coordinate ratio between the two methods (P＞0.05). It can be concluded that protein-chip reagent for detection of different HCV antibodies has higher sensitivity and specific than ELISA method and has the same accuracy as imported RIBA reagent. It will be a convenient and low costs reagent for diagnosis of HCV.

Abstract:The liver X receptors(LXR) nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism. After exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) respectively at different concentrations for 24 h, cholesterol efflux and liver X receptor α mRNA level were determined by FJ-2107P type liquid scintillator and reverse transcriptase-polymerase chain reaction(RT-PCR), respectively. 22(R)-hydroxycholesterol increases cholesterol efflux in THP-1 macrophage-derived foam cells with dose dependance and DIDS inhibits cholesterol efflux in THP-1 macrophage-derived foam cells with dose dependence too; RT-PCR showed that exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and DIDS at different concentrations for 24 h respectively, resulted in increase and decrease respectively in the expression of liver X receptor α mRNA in THP-1 macrophage-derived foam cells with dose dependence. It can be concluded that liver X receptor α plays an important role in cholesterol efflux in THP-1 macrophage-derived foam cells.

Abstract:A comparative analysis on the intron sequence oligonucleotide usages in two sets of yeast genes with higher and lower transcription frequencies, respectively, has shown that the intron sequence structures of the two sets of genes are different. There are more potential binding sites for transcription factors in the introns of the genes with high transcription frequencies. So it is speculated that introns regulate the transcription of genes. But more evidences are needed to favor this speculation. The detailed comparative analyses on the distribution (length and position) of introns and exons in the two sets of gene sequences also show that there is an obvious boundary between the lengths of the two sets of introns. There is no boundary between the lengths of the two sets of exons, although the means of their lengths are of discrepancy. The situation of the gene lengths (length of intron and exon) is similar to exon lengths. As far as the relative position, the introns in two sets of genes all have a bias toward the 5′ ends of genes. But as the actual position is considered, more introns in high transcription genes have a tendency to be located toward the 5′ ends of genes, some even located at 5′-UTR. These results suggest that the gene transcription rates are related to the length of intron, but not to the lengths of exons and genes sequences. The positions of introns may also influence the transcription rates. The transcriptional regulation of introns may be correlative with the transcriptional regulation of the upstream of genes, or be its continuous action.

Abstract:The mutation of tumor suppressor p53 gene probably produce p53 antibodies. p53 antibodies are of importance to tumor's diagnosis, prognosis and relapse monitor. Current method to detect p53 antibodies, i.e. enzyme-linked immunosorbent assay (ELISA), requires long time with multi-step, and the assay is only semi-quantitative. Immunomagnetic electrochemiluminescence (IM-ECL) assay was devoloped for quantitative detection of p53 antibodies in human serum. Results indicate that the detection limit of this assay is 10 ng/L. A stable calibration curve with a wide linear range from 0.01 to 1 000 μg/L was established, thus, made quantitation possible. Serum samples from lung cancer patients were tested using the IM-ECL assay. The assay uses only 50 μl of sample per test and requires a 30 min incubation period in addition to a 50 s acquisition time. The positive rate of p53 antibodies was 28.6% in lung cancer sera. Concentrations of p53 antibody in the positive carcinoma sera were quantified from the calibration curve. A trend was found that higher the p53 antibody concentration in the lung cancer serum likely linked to a higher diagonostic stage of the cancer. IM-ECL assay is significantly better in terms of detection limit, linear range, and assay time, compared to the similar paramenters of common ELISA. The results suggests that IM-ECL is a feasible method for rapid, sensitive and quantitative detection of p53 antibodies in human serum.

Abstract:Heterologous expression of recombinant proteins in yeast involves toilsome screening of a large number of recombinants from yeast cells. A quick and easy procedure was introduced for identifying recombinant clones from the yeast Saccharomyces cerevisiae by means of PCR without any prior DNA extraction and purification steps. This method involves a simple boiling step for 2～5 min of whole yeast cells suspended in water. Both the fresh and frozen competent S.cerevisiae cells can be used for transformation. The method of storing the competent yeast cells at －80℃ for future use is convenient and economic. A skill of decreasing the dimer products and increasing the specificity of PCR is recommended.

Abstract:To research the effect of insulin on the expression and activity of the neuronal nitric oxide synthase in neural cells, the effects of insulin on the expression and activity of neuronal nitric oxide synthase were investigated in PC12 cells, by using flow cytometry, in situ hybridization and electron spin resonance (ESR) techniques. After incubation with insulin for 9 h, the expression of nNOS protein was significantly increased by insulin in a concentration-dependent manner, with a maximal increase of about 55% of the control values (P＜0.01, n=3, t-test). The transcription of nNOS was also significantly increased by insulin (16 mU/L, 6 h), reaching （182±13）% of the control values (P＜0.01, n=3, t-test). In addition, significant increase of the activity of nNOS in PC12 cells was also detected after incubation with insulin (16 mU/L) for 9 h, with an increase of 67% of the control values (P＜0.01, n=4, t-test). It can be concluded that insulin can upregulate the expression and activity of neuronal nitric oxide synthase in PC12 cells.

Abstract:The rodent testes are generally more susceptible to cadmium(Cd)-induced toxicity than the liver. In order to clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, metallothionein(MT) gene expression, MT protein accumulation, and Cd retention were compared under different time in freshly isolated testicular sertoli cells and liver of rats treated with Cd. Adult male Sprague-Dawley rats received a s.c. injection of 4.0 μmol/kg and 0 h(control), 1 h, 3 h, 6 h, or 24 h later, tissue were sampled and testicular sertoli cells were isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. The results demonstrated that both isoform mRNA: MT1 and MT2 were increased after Cd treatment in liver and peaked at 3h, followed by a decline, in contrast, the mRNA levels in sertoli cells peaked at 6h. Cd exposure substantially increased hepatic MT, but did not increase MT translation in sertoli cells. These results indicate that: (1)metallothionein mRNA expression is tissue- and time-dependent; (2) the inability to induce the metal-detoxicating MT-protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.

Abstract:To obtain recombinant sonic hedgehog N-terminal protein (Shh-N)and study its biological activity，the cDNA encoding the functional rat Shh-N was isolated using PCR. The expression plasmid was constructed by inserting Shh-N cDNA into plasmid pET28a (+) and transformed into E.coli. Then an expression strain was selected. SDS-PAGE and Western blotting analysis revealed that the rat Shh-N protein was highly expressed in the form of a large quantity of soluble protein and a small amount of inclusion body being induced by the IPTG. After Ni-NTA resin affinity chromatography, the purity of the final Shh-N protein was more than 85%. Purified protein being added to neural progenitor cells during developmental stages could induce the phenotype of tyrosine hydroxylase-immunopositive neurons. Based on this study, sufficient donor cells would be available to treat Parkinson′s disease in clinical therapy via inducing different kinds of stem cells from human being into dopaminergic neurons.

Abstract:With many genomes completed and extensive applications of DNA chips, analysis of the gene expression data has become a hotspot in the postgenomic age. Clustering is the art to group genes with related functions according to the similarities in their expression profiles. A number of clustering algorithms have been developed for gene expression data analysis. For their respective focuses and principles, every method has its own advantages and disadvantages, which are reviewed. How to evaluate the capabilities of these algorithms, and to develop new methods more suitable for gene expression analysis, should be urgent.

Abstract:Protein-protein interactions play a central role in numerous processes in the cell. Given the complex organization of cell, it is very likely that the molecular behavior of proteins in a test tube is different from that in living cells. For this reason, the conventional approaches used to study protein-protein interactions, including two-hybrid yeast assay, immunoprecipitation assay, etc, may not represent the physiological nature in living cells. Fluorescence resonance energy transfer (FRET) is a recently developed laser technique, which offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes within a living cell and to follow the time course of the changes in FRET corresponding to cellular events. The observation of such dynamic molecular events in its natural environment provides vital insight into the protein-protein interactions within the cell. The applications of this approach to study the protein-protein interactions in vivo are briefly reviewed.