Abstract:鼻咽癌是一种多基因参与、遗传因素与环境因素协同作用的恶性肿瘤，具有明显的民族聚集性和地域分布特征．鼻咽癌在中国南方以及国外的中国南方移民和后裔中发病率极高，中国广东省的发病率明显高于欧美等其他地区25至30倍，因此被称为“中国癌”．这种惊人的地域性和民族性特征使鼻咽癌的防、诊、治在中国癌症研究中具有特殊重要的位置．迄今，尽管有很多的研究，对鼻咽癌的发病机制仍不十分清楚，环境因素难于把握，其治疗效果不能令人满意，五年生存率仍徘徊在50%左右．因此，寻找鼻咽癌发生、发展和预后相关的分子标志物，推动实现早期诊断和个体化治疗，成为一个重要的研究方向．本期发表的《 鼻咽癌分子标志物研究 》(见7～13页)系统总结了中南大学肿瘤研究所李桂源教授领导的研究集体在这一领域已经取得的研究成果．他们运用基因组学、转录组学、蛋白质组学技术和组织微阵列等高通量技术，对不同阶段、不同组织类型和不同临床分期的鼻咽标本进行了大规模筛选，在大样本基础上构建了鼻咽癌不同发展阶段的分子靶标系统，发现相应的分子标志物，不仅为鼻咽癌分子分型研究奠定了重要工作基础，而且对推动鼻咽癌的早期诊断和个体化治疗有重要意义．
多阶段性是鼻咽癌发生和发展的重要特征，从正常的鼻咽黏膜被覆上皮和陷窝柱状上皮演变到异型增生(癌前病变)、早期浸润癌、中晚期浸润癌和转移癌，需要经过一系列不同的发病阶段和病理阶段，涉及多种分子事件．根据这一特点，李桂源研究组对各阶段的分子标志物进行了全方位的研究，在鼻咽癌早期发生、侵袭转移、预后、复发等各重要环节都鉴定了一系列可用于诊治参照的靶标和候选分子标志物．特别是，发现鼻咽癌早期诊断的理想分子靶标 (EBER-1，p16，p27，SPLUNC1) 具有重要价值．因为鼻咽癌发病部位隐蔽，特别是在咽隐窝及鼻咽顶壁处，早期症状不明显，寻找早期诊断的特异分子标志物是实现鼻咽癌早期诊断的紧迫课题，意义重要．同时，他们通过定位克隆策略，结合cDNA和组织微阵列技术途径，筛选到鼻咽癌侵袭与转移预测的分子靶标(NGX6，LTF，Ezrin等)．鼻咽癌转移是一个非常复杂的过程，涉及基因不稳定性和基因变异的积累，以及激活的癌基因与抑癌基因的失衡，他们的研究结果是这方面的重要进展．这些结果与鼻咽癌预后相关、复发相关以及放、化疗敏感度相关的分子标志物的筛选与鉴定相结合，共同构成了一个较全面的分子标志物研究格局，为今后在这一重要课题上进行更深入更有成效的研究建立了很好的基础．
鼻咽癌的发生与发展涉及了多个癌基因和抑癌基因的共同参与，它们的激活、失活及相互作用构成了鼻咽癌病理过程的主要分子基础，寻找可有效用于临床的各个环节的分子标志物还是一个有待解决的重要课题．正如该文作者在文中指出的，目前对这些分子标志物的研究从总体上还处于初步探索阶段，需要更系统深入地研究作用于鼻咽癌发病不同阶段的生物靶点基因，并在大样本的组织中进行验证，在临床试验中进行有效评估，以推动实现鼻咽癌早期诊断和个体化治疗的进程．

Abstract:Nasopharyngeal carcinoma (NPC) is a malignant disease which had critically threatened human?蒺s health. It is very important to look for the biomarkers related to early diagnosis and prognosis of NPC. Using a series of large scale screening techniques from genomics, transcriptomics, proteomics and tissue microarray, combined with some international progress, a molecular marker system had been preliminarily constructed in different stages of NPC: (1) SPLUNC1, EBER-1, p16, p27, RASSF1A and CDH13, a group of molecular targets for the early diagnosis of NPC. (2) The class forecast model consisting of up-regulated RB1, STMN1, DSP and down-regulating SERPINB6, AGTRL1, SYTL2, the biomarkers to identify between normal nasopharyngeal epithelium and NPC. (3) NGX6, Ezrin, LTF, OPN, THY1 and Tiam-1, a group of candidate biomarkers forcasting the invasion and metastasis of NPC. (4) Cyclin D1，Survivin and HPA, a group of biomarkers related to the prognosis of NPC. (5) Bcl-2、EGFR and Ki67 proteins, a group of perfect candidate biomarkers for forecasting radiation sensitivity of NPC. (6) SAA and cox-2, the candidate biomarkers monitoring NPC recurrence. (7) The changes of six SNP from BRD7, NGX6, NOR1 and UBAP1, a group of the inheritance susceptibility risk factors. (8) Constructing a biomarker system of different clinical stages of NPC composed of 139 genes. These biomarkers based on screening with large amounts of samples lay an important foundation for molecular typing research of NPC.

Abstract:Ubiquitination on target proteins is the signal of cellular protein degradation. Ubiquitin ligase E3 is one of the key enzymes in ubiquitination, it recognizes a specific substrate protein and recruits an ubiquitin conjugating enzyme E2, mediating the ubquitin transfer from the E2 to the substrate protein. Ubiquitin ligase E3 can be divided into two subfamilies according to their different structure characters and function mechanisms, the HECT (homologous to E6AP C terminus) family and the RING-finger family. Members of the HECT E3 share the common HECT catalytic domain, which can bind to an E2 and load the ubiquitin on themselves before catalyzing the transfer of ubiquitin to the target proteins. While the RING-finger E3 all contain an similar E2 binding domain and a unique substrate binding part, mediating direct ubiquitin transfer from the E2 to the substrate. The most recent progresses in the stuctural and functional studies of these two E3 famlies were summarized.

Abstract:Being organelles present in higher plants and algae, and containing the entire machinery for the process of photosynthesis and their own genetic system, chloroplasts have played important roles in plant evolution. With the help of molecular approaches, the structure and sequences of chloroplast genomes have been studied to a large extent. The valuable chloroplast genomic information has been utilized in investigating plant evolution, diversity and phylogenetic relationships between different species. At the same time, chloroplast genome sequences are the essential information paving the road for successful chloroplast genetic engineering, which has shown considerable advantages compared with nuclear transformation and demonstrated its great potential in genetic improvement and the production of biopharmaceuticals. The latest developments in chloroplast genome analysis are reviewed, providing readers with insight into the fast growing field and facilitating a comprehensive understanding of the evolution, genetics and phylogenetic relationships among plants. It is also hoped to promote the further development of chloroplast technology and the commercial application of chloroplast transformation.

Abstract:NOK is a newly identified receptor protein-tyrosine kinase (PRTK) molecule that can promote tumorigenesis and metastasis. Previous data showed that NOK could activate the phosphatidylinositol 3'-kinase (PI3K) pathway in stable BaF3 cells. But how does NOK activate PI3K in cells remains unknown. It was showed that NOK physically interacted with the PI3K downstream effector Akt and enhanced its activation in human embryo kidney 293T (HEK293T) cells. Deletion mapping indicated that protein kinase B (Akt) was able to directly contact with the kinase domain of NOK. Inactivating the Akt kinase domain significantly reduced the intermolecular interaction between NOK and Akt, while constitutively active mutant of Akt apparently had a stronger interaction with NOK. NOK did not have an additive effect on insulin-mediated Akt activation. Overall, the results indicate that NOK might complex with Akt and directly activate PI3K/Akt signaling pathway.

Abstract:Collaboration networks have proven informative when used to describe various kinds of human relationships. Similar strategy could be used in the transcriptional regulatory network. A collaboration network of target genes (TGs) was constructed based on common transcription factors (TFs), and similarly, a smaller network of transcription factors was constructed based on their common target genes. After clustering the target gene collaboration networks, genes in the same cluster were often enriched for one or more GO terms. The results also showed that genes with specific GO terms tend to share similar regulatory mechanisms. It indicates that in a collaboration network approach the relatively simple "regulatory mechanism" measure used here was able to extract considerable biologically relevant information. Moreover, a definition of anomaly used before in a bipartite graph analysis method was applied into the collaboration networks analysis. And the correlation between the anomalies and the essential genes was discovered. In a conclusion, a collaboration network approach may be a valuable supplement to other analyses of transcriptional networks.

Abstract:Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. Sef has been reported to function in different ways, however the regulation of Sef stability remains unknown. The possible role of c-Cbl in the regulation of Sef protein degradation was investigated. Results from coimmunoprecipitation and immunostaining assays reveal that hSef colocalizes and interacts with c-Cbl. Data suggest that the interaction between hSef and c-Cbl results in the ubiquitination and subsequent degradation of the hSef protein. It was proposed that c-Cbl may serve as a modulator to regulate Sef protein stability during FGF signal transduction.

Abstract:DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is specific receptor on Dendritic cells, and plays a pivotal role on antigens presentation. Uptodate, the clear regulation mechanisms for DC-SIGN expression are not available. IL-4 is one of the most important cytokines inducing DC-SIGN production, while, NF-κB is an important transcription factor controlling signaling transduction. Both IL-4 and NF-κB are closely related to DC-SIGN regulation. NF-κB and IL-4 actions on DC-SIGN promoter activity, DC-SIGN expression as well as interactions between IL-4 and NF-κB were investigated in THP-1 cell. It was found that the mutation of NF-κB binding site in DC-SIGN promoter results in DC-SIGN promoter activity decrease about 50%. NF-κBp50 stimulates DC-SIGN expression in THP-1 cells. IL-4 upregulates DC-SIGN expression on THP-1 cells as well as NF-κB production. These data reveal that NF-κB is associated with IL-4 induced DC-SIGN expression.

Abstract:Chlamydial infection in human urogenital tract induces inflammation and causes tissue damage and scarring. It is thought that cytokine production by the Chlamydia-infected cells plays a key role in chlamydial disease processes. Although many cytokines have been detected during chlamydial infection, little is known about the molecular mechanisms on how Chlamydia triggers and sustains the inflammatory cytokine cascades. In the current study, chlamydial infection of the human cervical epithelial cell line HeLa cells can induce the production of IL-8, IL-1α, IL-1β and IL-6. Using inhibitors for probing intracellular kinase signaling pathways required for the Chlamydia-induced cytokines, it was found that the Chlamydia-activated MAPK / P38 pathway is required for the chlamydial induction of IL-1α and IL-6 while both the Chlamydia-activated MAPK/ERK and MAPK/P38 pathways contribute to the production of IL-8.

Abstract:The cDNA of a human Mini-proinsulin (B¹-B²⁹)-AAK-(A¹-A²¹) which named HMPIDesB³⁰ (human mini-proinsulin des B³⁰) was synthesized and inserted into the Escherichia coli-yeast shuttle vector pPIC9K. The constructed plasmid, HMPIDesB³⁰/pPIC9K was transformed into the GS115 cells of the methylotrophic yeast, Pichia pastoris, using electrotransformation. A transformant with a high copy number of the gene integrated into the chromosome was obtained by YPD plates which contains increasing concentrations of G418. After fed-batch fermentation, the protein was purified by macroporous resin, ion-exchange chromatography and precipitated with 10～20 mmol/L Zn²⁺. The purity of HMPIDesB³⁰ is 95%，the expression level reached 1.0 g/L, and the recovery rate of purification is about 60%. The purified protein was characterized by 16.5% Tricine SDS-PAGE, HPLC, and mass spectrometry, and the molecular mass of the expressed products is in accordance with the cumulated value. HMPIDesB³⁰ can be secretorily expressed by Pichia pastrois. The composition of the fermentation medium, the optimal condition of the fermentation and an effective method to purify the expression products from the culture were established.

Abstract:Melanin is a kind of biopolymer, composed of a complex quinine/indole-quinone derived mixture which is produced in melanocytes from tyrosine. More than 100 genes related to melanin deposition have been found in mouse. Slc24a5 (solute carrier family 24 , member 5) is a novel gene first cloned in zebrafish. And it has been confirmed that the Slc24a5 is involved in controlling the melanin deposition in zebrafish. Here the full length of the chicken slc24a5 mRNA sequence, its expression profile and a discussion of its relationship to melanin deposition in chicken were reported. Chicken Slc254a5 has a CDS of 1 269bp, predicting a protein of 423 amino acids which is about 80 amino acids shorter than in human and mouse. It has 9 exons and 8 introns and spans more than 11kb of genome sequence. The quantitative real-time PCR confirmed that the chicken Slc24a5 was highly expressed in the eye of White Leghorn and Beijing Fatty Chickens, and in the eye, skin and muscle of Silky. The relative expression in Silky eye is more than two times that of White Leghorn. The relative expression in Silky skin is about 70 times that of White Leghorn and about 15 times that of Beijing Fatty Chickens. The relative expression in Silky muscle is about 15 times that of White Leghorn and about 3 times that of Beijing Fatty Chickens. And the expression result is in accordance with the melanocyte and melanin deposition in these tissues.

Abstract:Acidithiobacillus ferrooxidans is a chemolithotrophic microorganism capable of using ferrous ions and sulphides as energy sources. This microorganism has an important role in the bioleaching of minerals. During this process, the bacteria are normally subjected to several stressing conditions, such as temperature changes, lack of nutrients or pH changes, which may affect the efficiency of the bacterial action. SELDI is a recent technology that allows for high-throughput proteomics studies. The Protein Chip SELDI technology was used to generate comparative protein profiles of Acidithiobacillus ferrooxidans grown under phosphate starvation or normal condition additionally adding Fe²⁺ as energy resource. There were 13 significantly differential expressed protein's peaks found by using SELDI Protein Chip technologies, which made a solid foundation for further isolation these low molecular proteins by adopting technologies such as HPLC etc.

Abstract:Chan-Su(CS, or bufonis venenum), a traditional Chinese medicine, has many biological functions. It is mainly composed of bufotenidines, bufogenins, and etc. CS has been documented to possess functions against inflammation and cancer, and is widely employed as a therapeutic drug for many kinds of cancers in China. However, it is difficult to judge antitumor effect of agents derived from CS because of the following reasons: agents derived from CS are mixture, they are lack of observations of multiple centres, preparation process is lack of quality control standards and agents have many toxic or side effects at high dose. Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. The mechanisms of the initiation and regulation of apoptosis are complex and diverse. Caspase family is closely connected with many apoptotic processes, while its member, caspase-3, being an important executive apoptotic molecule. To explore the inhibitory effect and mechanism of CS on human lung adenocarcinoma (ASTC-a-1), gene plasmid transfection, fluorescence emission spectra and fluorescence resonance energy transfer (FRET) were used to study the caspase-3 activation during the CS-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis．CCK-8 was used to assay the inhibition of CS on the cells viability. The dynamical emission spectra of SCAT3 were performed inside living cells expressed stably with SCAT3 after CS treatment. The cell morphology was examined and photographed by phase microscope and confocal fluorescence scanning microscope．Experimental results showed that (1) CS inhibited dose-dependently the cells viability; (2) apoptotic body was observed in some cells 6 h after CS treatment, and most of cells were in shrinkage 24 h after CS treatment; (3) The SCAT3 inside living cells were cleaved by caspase-3 2 h after CS treatment, and most of the SCAT3 was cleaved 24 h after CS treatment, and the acceptor photobleaching experiments of SCAT3 also verified these results, implying that CS activated caspase-3 which induced cell apoptosis. Therefore, caspase-3 is involved in the CS-induced cell apoptosis.

Abstract:Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction, which includes an N-terminal actin-binding domain, central catalytic domain, calmodulin-binding domain, and a C- terminal myosin-binding domain. Myosin phosphorylated by the catalytic domain of MLCK is in an active form and interacts with the actin filament to contract smooth muscle. This mode of phosphorylation is widely accepted as the regulatory mechanism for actin-myosin interaction. However, there are a number of observations that are not explained by this mechanism. An MLCK C-terminal fragment (MLCK fragment) containing the myosin-binding domain have been previously engineered but devoid of a catalytic domain, which has confirmed how myosin is stimulated by this non-kinase pathway. A recombinant GST-fusion protein of the MLCK fragment was expressed in E.coli and detected by SDS-PAGE. Through Glutathione-Sepharose 4B affinity chromatography, a single pure band of the MLCK fragment was obtained. The phosphorylated myosin ATPase activities of the MLCK fragment, as well as its proteolytic fragment HMM and S1 were measured with the EnzChek Phosphate Assay Kit. The MLCK fragment stimulated phosphorylated myosin ATPase activity (V_max=(19.426±1.669)-fold, K_m=(0.486±0.106)μmol/L). Similar stimulation figures were obtained by measuring the ATPase activity of phosphorylated HMM and S1. The data suggests that MLCK could activate phosphorylated myosin, HMM and S1. In addition, in vitro motility assays demonstrated that increasing amounts of the MLCK fragment increased actin-myosin interaction and sliding. Also, the velocity of actin filaments could be enhanced on a gizzard smooth muscle myosin surface with the MLCK fragment. It is conclude that the non-kinase C-terminal domain of MLCK is independent of the phosphorylating mode for activation of myosin.

Abstract:Despite the clinical and experimental concerns about the deleterious effects of neonatal seizures on brain development, the underlying mechanism of seizure-induced brain damage is still not clear. Moreover, early therapeutic intervention studies are also less available. For this reason, the study was performed to explore the long-term effects of neonatal seizures and physical exercise on learning, memory and the expression of calcium/calmodulin- dependent protein kinase Ⅱ (CaMKⅡ). Twelve neonatal rats for each group were assigned: the single- seizure group (SS), the recurrent-seizure group (RS) and the control group. The volatile agent flurothyl was used to induce 30 min seizure attack. At postnatal day 6(P6), the single seizures induced only once and recurrent seizures induced once per day for consecutive 6 days. Control rats were placed into the container for an equal amount of time to their counterpart without exposure to flurothyl. Morris water-maze test were performed at P27～P31, P58～P61 and P80～P82, meanwhile at P51～P56, the RS and SS groups were submitted to forced running exercise. In situ hybridization method was used to detect the expression of CaMKⅡ mRNA in hippocampus. The results are as follows: (1) Escape latency. In the first two Morris water-maze tests, there was a decreasing trend of escape latency in three groups, and the escape latency of RS group was much longer than that of control group. After physical exercise,in the last Morris water-maze tests, the diference of escape latency in three groups is not significant. (2) Searching strategy. In the first Morris water-maze test, there was a decreasing trend of marginal strategy and an increasing trend of taxis strategy in three groups, but the frequency of marginal strategy was higher and the frequency of taxis strategy was lower in RS group than that in SS and control group in the third and fourth day（P < 0.01）. However, there were no significant differences among three groups in the second and third Morris water-maze test. (3) Memory test. In the three memory tests, as for the distance ratioin between the origin platform quadrant to total distance, the RS group was the worst. The frequency of taxis strategy was much lower in RS group than that in SS and control groups in the first to third day(P < 0.01). (4) In situ hybridization detection showed that the expression of CaMKⅡ mRNA in dentate gyrus and hilus was much lower in RS group than that in SS and control groups (P < 0.01). It was concluded that recurrent but not single prolonged seizures could cause long-term effects on learning and memory, which may be associated with the down-regulated expression of CaMKⅡ mRNA in hippocampus. Physical exercise improved the learning capacity of RS group but with no effect on memory capacity of RS group.