A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium

Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).     

Homology modeling provides insights into the binding mode of the PAAD/DAPIN/pyrin domain,a fourth member of the CARD/DD/DED domain family

The PAAD/DAPIN/pyrin domain is the fourth member of the death domain superfamily, but unlike other members of this family, it is involved not only in apoptosis but also in innate immunity and several other processes. We have identified 40 PAAD domain-containing proteins by extensively searching the genomes of higher eukaryotes and viruses. Phylogenetic analyses suggest that there are five categories of PAAD domains that correlate with the domain architecture of the entire proteins. Homology models built on CARD and DD structures identified functionally important residues by studying conservation patterns on the surface of the models. Surface maps of each subfamily show different distributions of these residues, suggesting that domains from different subfamilies do not interact with each other, forming independent regulatory networks. Helix3 of PAAD is predicted to be critical for dimerization. Multiple alignment analysis and modeling suggest that it may be partly disordered, following a new paradigm for interaction proteins that are stabilized by protein-protein interactions.     

电喷雾离子化/质谱法(ESI/MS)及其在生物大分子研究中的应用

电喷雾离子化／质谱法（ESI／MS）在各种有机化合物、多肽、蛋白质（含糖蛋白）、核苷酸、糖、脂类及合成高分子物质等分析领域获得了广泛的应用,本文系统介绍了ESI／MS的基本原理,其联用技术,及其在生物大分子研究,包括肽图谱测定,糖分析和核苷酸分析中的应用。     

The COP/DET/FUS proteins-regulators of eukaryotic growth and development

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.     

Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization

The biological activity of a lectin is influenced by its quaternary structure. Viscumin is special among the family members of toxic AB-type plant lectins, because it triggers mitogenicity, toxicity, and agglutination. Its activity profile is dependent on the concentration, motivating a thorough inspection of the status of quaternary structure. Over a broad range of protein concentrations (0.01-25 mg/mL), viscumin occurs as a dimer. At high concentrations, the solutions exhibited nonideality, self-association, and polydispersity in sedimentation equilibrium and velocity experiments caused by irreversible aggregation. Calculation of viscumin's overall shape based on sedimentation velocity data resulted in an elongated dimer form resembling that of crystallized agglutinin. Appearance of monomers was restricted to concentrations in the submicrogram/mL level, as demonstrated by fast protein liquid chromatography gel-filtration analysis. To shift the equilibrium to the monomer for comparative cell biological assays, we performed chemical modification under conditions protecting the lectin activity. Citraconylation was effective to destabilize the dimer. Binding studies by fluorescence-activated cell scan analysis revealed a reduction in cell association upon modification and a tendency for increased sensitivity towards haptenic inhibitors at microg/mL concentrations. Nonetheless, growth inhibition continued to be potent for the ricin-like monomer despite reduced extent of binding. Occurrence of a concentration-dependent monomer/dimer equilibrium appears to achieve the same objectives as the development of two separate protein entities in Ricinus communis, an alternative strategy to emergence of a monomeric toxin, and cell cross-linking dimeric agglutinin.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.     

Role of regulatory miRNAs of the PI3K/AKT/mTOR signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the common malignant human tumors with high morbidity worldwide. Aberrant activation of the oncogenic phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling is related to clinicopathological features of HCC. Emerging data revealed that microRNAs (miRNAs) have prominent implications for regulating cellular proliferation, differentiation, apoptosis, and metabolism through targeting the PI3K/AKT/mTOR signaling axis. The recognition of the crucial role of miRNAs in hepatocarcinogenesis represents a promising area to identify novel anticancer therapeutics for HCC. The present study summarizes the major findings about the regulatory role of miRNAs in the PI3K/AKT/mTOR pathway in the pathogenesis of HCC.     

Inhibition of PLK4 might enhance the anti-tumour effect of bortezomib on glioblastoma via PTEN/PI3K/AKT/mTOR signalling pathway

Glioblastoma (GBM) is one of the most common aggressive cancers of the central nervous system in adults with a high mortality rate. Bortezomib is a boronic acid–based potent proteasome inhibitor that has been actively studied for its anti-tumour effects through inhibition of the proteasome. The proteasome is a key component of the ubiquitin-proteasome pathway that is critical for protein homeostasis, regulation of cellular growth, and apoptosis. Overexpression of polo-like kinase 4 (PLK4) is commonly reported in tumour cells and increases their invasive and metastatic abilities. In this study, we established a cell model of PLK4 knockdown and overexpression in LN-18, A172 and LN-229 cells and found that knockdown of PLK4 expression enhanced the anti-tumour effect of bortezomib. We further found that this effect may be mediated by the PTEN/PI3K/AKT/mTOR signalling pathway and that the apoptotic and oxidative stress processes were activated, while the expression of matrix metalloproteinases (MMPs) was down-regulated. Similar phenomenon was observed using in vitro experiments. Thus, we speculate that PLK4 inhibition may be a new therapeutic strategy for GBM.     

PI3K/AKT/mTOR信号通路及其在卵巢癌治疗中的应用

卵巢癌是女性生殖系统常见的恶性肿瘤,发病率居于妇科恶性肿瘤第三位,死亡率居于妇科恶性肿瘤之首。目前对卵巢癌的标准治疗包括肿瘤细胞减灭术及卡铂和紫杉醇的联合化疗。PI3K/AKT/mTOR信号通路在卵巢癌的细胞增殖、侵袭、细胞周期进展、血管生成及耐药中发挥重要作用,是卵巢癌中最常发生改变的细胞内途径。本文对PI3K/AKT/mTOR信号通路及其在卵巢癌增殖和进展中的影响、PI3K/AKT/mTOR信号通路抑制剂在卵巢癌中的治疗应用做简要综述。     

Thermodynamics and stability of the PAAD/DAPIN/PYRIN domain of IFI-16

The PAAD domain is a conserved domain recently identified in more than 35 human proteins that are involved in apoptosis and inflammatory signaling pathways. Structural studies have confirmed that this domain belongs to the death domain superfamily which includes PAAD/CARD/DED/DD families. Recently, the 3D structures determined by NMR of NALP1 and ASC PAAD domain, members of the PAAD family, have shown that it is composed of a 6 helix bundle as with other death domain family members. However, helix-3 in the solved structures is unordered in solution. In this study we compare the thermodynamic, folding and stability properties of different members of the PAAD and CARD families and investigate structural conformational changes induced by the helix inducers trifluoroethanol and SDS on the PAAD domain of IFI16 and on the CARD domain of RAIDD. We show that inside the PAAD and CARD families, members have similar thermodynamic properties, however, the DeltaG of folding for PAAD and CARD members are, respectively, -1.4 and -5.5 kcal mol(-1). This difference is attributed to less alpha helical content for PAAD due to the unfolding of helix-3 that lowers bonded energy and increases disorder when compared to CARD members. Despite identical fold between PAAD and CARD families but limited sequence identity, there are striking differences in the thermodynamics of both families.     

SSeCKS/Gravin/AKAP12 Inhibits Cancer Cell Invasiveness and Chemotaxis by Suppressing a Protein Kinase C- Raf/MEK/ERK Pathway

SSeCKS/Gravin/AKAP12 (“SSeCKS”) encodes a cytoskeletal protein that regulates G₁ → S progression by scaffolding cyclins, protein kinase C (PKC) and PKA. SSeCKS is down-regulated in many tumor types including prostate, and when re-expressed in MAT-LyLu (MLL) prostate cancer cells, SSeCKS selectively inhibits metastasis by suppressing neovascularization at distal sites, correlating with its ability to down-regulate proangiogenic genes including Vegfa. However, the forced re-expression of VEGF only rescues partial lung metastasis formation. Here, we show that SSeCKS potently inhibits chemotaxis and Matrigel invasion, motility parameters contributing to metastasis formation. SSeCKS suppressed serum-induced activation of the Raf/MEK/ERK pathway, resulting in down-regulation of matrix metalloproteinase-2 expression. In contrast, SSeCKS had no effect on serum-induced phosphorylation of the Src substrate, Shc, in agreement with our previous data that SSeCKS does not inhibit Src kinase activity in cells. Invasiveness and chemotaxis could be restored by the forced expression of constitutively active MEK1, MEK2, ERK1, or PKCα. SSeCKS suppressed phorbol ester-induced ERK1/2 activity only if it encoded its PKC binding domain (amino acids 553–900), suggesting that SSeCKS attenuates ERK activation through a direct scaffolding of conventional and/or novel PKC isozymes. Finally, control of MLL invasiveness by SSeCKS is influenced by the actin cytoskeleton: the ability of SSeCKS to inhibit podosome formation is unaffected by cytochalasin D or jasplakinolide, whereas its ability to inhibit MEK1/2 and ERK1/2 activation is nullified by jasplakinolide. Our findings suggest that SSeCKS suppresses metastatic motility by disengaging activated Src and then inhibiting the PKC-Raf/MEK/ERK pathways controlling matrix metalloproteinase-2 expression and podosome formation.     

A/California/07/2009亚型猪流感冷适应减毒疫苗株的拯救及免疫效果评价

应用反向遗传学技术,选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60 ca (H2N2)型流感病毒的6个内部基因为骨架,与A/California/07/2009株流感病毒2个抗原基因HA、NA分别克隆到polⅠ-polⅡ转录表达载体pAD3000中,构建8个转录表达载体重组质粒,共转染Vero细胞,获得重配A/California/07/2009ca株流感病毒．重配病毒的TCID₅₀为7.5,病毒传4代后其血凝素(HA)滴度稳定在1∶256,半数感染剂量EID₅₀为8,鸡胚传20代,经RT-PCR鉴定未发现重组病毒基因突变,电镜观察重配病毒符合流感病毒的主要特征;蔗糖纯化的病毒经肌肉注射(灭活)及滴鼻(减毒活病毒)两种途径免疫BALB/c小鼠,结果显示：滴鼻免疫和肌肉注射都可以产生较高效价的血凝抑制(HI)抗体,肌肉注射组产生的HI抗体略高(P = 0.044),但肌肉注射组检测不到高效价IgA抗体;滴鼻免疫组鼻冲洗液中可以检测到高效价的IgA抗体,同型病毒感染后,IL-1β、TNFα、IFN-α等前炎因子分泌较早,且高于肌肉注射组(P < 0.05),可见,喷鼻减毒疫苗比灭活全病毒疫苗能更好地激发黏膜免疫反应．通过对小鼠各个器官病毒载量的检测发现,4天后鼻腔、气管、脑、肺、脾脏没有病毒存在,证明减毒活疫苗株在小鼠上是安全的．以上数据可以初步断定,重组病毒有作疫苗候选株的可能,而且喷鼻疫苗具有降低免疫剂量、同时激活体内体液免疫和细胞免疫的功能．