18F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model

Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-¹⁸F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide (¹⁸F-FBHGal), a new ¹⁸F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5 μM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of ¹⁸F-FBHGal in fibrosis liver (14.84 ± 1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50 ± 1.51 %ID/g, P <0.01) at 30 min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30 min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T_1/2α, T_1/2β, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of ¹⁸F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that ¹⁸F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.     

四氯化碳诱导小鼠肝脏纤维化的差异蛋白质组学

以四氯化碳(Carbon tetrachloride,CCl4)诱导小鼠肝组织纤维化为研究模型,发现并探讨纤维化肝组织与正常肝组织在蛋白质组水平上的差异。实验小鼠(C57 BL/6)随机分为两组,由橄榄油和四氯化碳诱导15周,并分别对这两组肝组织的全蛋白表达谱进行质谱检测,应用GO(Gene Ontology)功能分类分析和KEGG(Kyoto Enyoolpedia of Genes and Genomes)信号通路的富集分析方法对鉴定到的全蛋白表达谱进行差异表达分析。在对照组和实验组中,我们分别鉴定到17 382和20 486条特异性肽段,图谱平均利用率大于50%,共计鉴定到蛋白4 991种(蛋白特异性肽段个数至少为1),其中差异表达蛋白有2 135种(差异倍数大于或等于2),表达上调蛋白1 264种,下调蛋白871种。纤维化肝脏组织中与细胞外基质组成(Extracellular matrix organization)、细胞骨架组成(Cytoskeleton organization)、有机磷酸代谢(Organophosphate metabolic process)、细胞定位(Cellular localization)和细胞组分调节(Regulation of cellular component organization)相关蛋白的表达是上调的;另外,与小分子代谢(Small molecule metabolic process)、蛋白质转运(Protein transport)和有机氮化合物的代谢(Organonitrogen compound metabolic process),以及四吡咯的合成过程(Tetrapyrrole biosynthetic process)有关蛋白的表达是下调的。信号通路富集分析结果表明,纤维化与VEGF和T细胞受体信号调节通路密切相关。结果提示,纤维化的形成不仅是一个复杂的信号转导过程,更是一个炎症与免疫相互促成的结果;增强肝实质细胞的存活,降低相关信号的传递及接收都有可能对纤维化的发生和发展起到抑制效果。     

骨髓移植小鼠二甲基亚硝胺肝纤维化模型的建立

目的构建绿色荧光蛋白(GFP)标记骨髓细胞的小鼠,并复制其二甲基亚硝胺(DMN)肝纤维化模型。方法 ICR雄性小鼠32只,随机分为正常组6只和移植组26只。移植组接受致死量γ射线照射后,经尾静脉输入GFP转基因小鼠的骨髓细胞;正常组不进行照射和移植,仅尾静脉注射等量生理盐水。两个月后制备血涂片,观察移植组造血重建情况,造血重建动物再分为对照组和造模组,造模组用DMN按每次10mg/kg体重腹腔注射制备肝纤维化模型,隔日一次,正常组和对照组给予等量生理盐水。设染毒后3周和4周两个时间观察点,生化法测定肝功能;Jamall法检测肝组织羟脯氨酸含量;HE染色及天狼猩红染色观察肝组织炎症、坏死及纤维组织增生情况;GFP免疫荧光组织化学观察骨髓源性细胞在肝脏的归巢特点。结果骨髓移植两个月后,移植组外周血中出现满视野GFP+细胞。与正常组比较,两个时间观察点造模组肝功能(ALT、AST、Alb及T.Bil)均明显异常(P<0.05),肝组织羟脯氨酸含量显著增高(P<0.05),造模3周末肝组织出血性坏死,有炎性细胞浸润,但尚未形成完全的纤维间隔;造模4周末肝组织炎症、坏死程度加重,可见完全的纤维间隔,在DMN造模动物肝组...     

Changes in hepatic protein expression in spontaneously hypertensive rats suggest early stages of non-alcoholic fatty liver disease

Hypertension is a systemic disorder affecting numerous physiological processes throughout the body. As non-alcoholic fatty liver disorder (NAFLD) is a common comorbidity of hypertension in humans, we hypothesized that molecular hepatic physiology would be altered in a model of genetic hypertension. Despite the broad use of the spontaneously hypertensive rat (SHR) model, little is known regarding how hypertension influences hepatic function under basal conditions. In order to determine whether hypertension induces changes in the hepatic protein expression suggestive of early stages of NAFLD, we compared the whole tissue proteome of livers from SHR and Wistar Kyoto (WKY) 16 week old rats using 2DGE and MALDI-TOF MS. Fifteen proteins were identified that display different levels of expression between the SHR and WKY livers: 50% of proteins have mitochondrial or anti-oxidant functions while 20% are involved in lipid metabolism. Quininoid dihydropterin reductase, sulfite oxidase, and glutathione-S-transferase mu 1 were all identified as either undergoing a difference in post-translation modification or a difference in protein abundance in SHR compared to WKY livers. As oxidative stress is a well described component of both NAFLD and hypertension in SHR, the identification of novel changes in protein expression provides possible mechanisms connecting these two pathologies in humans.     

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.     

大鼠急性低氧模型尿液蛋白质组的变化

本研究旨在探究急性低氧对大鼠尿液蛋白质组造成的影响。在该项研究中,大鼠被放置于模拟海拔5 000 m高原环境的低氧舱内24 h。在低氧后0、12、24 h收集尿液样本,并使用液相色谱-串联质谱技术（LC-MS/MS）对尿蛋白进行分析。与低氧0 h相比,低氧12 h组共鉴定到144个差异蛋白,低氧24 h组共鉴定到129个差异蛋白。功能分析显示,差异蛋白参与了一系列与低氧应激有关的生物学通路,如抗氧化应激、糖酵解、补体和凝血级联反应等。研究结果表明,尿液蛋白质组可以反映急性低氧刺激后的显著变化。这些发现可能提供了一种判断机体缺氧状态的方法,有助于辅助检测缺氧状态。     

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

Background  

Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry.     

Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development

The patterns of protein synthesis in teratocarcinoma stem cells (embryonal carcinoma cells) and in mouse embryos at various stages of preimplantation development were studied using SDS-polyacrylamide slab gel electrophoresis with autoradiography. Significant differences were observed in comparisons of embryonal carcinoma cells with isolated inner cell masses (ICMs) or with embryonic cells at earlier stages of development. However, no such differences in the overall pattern of protein synthesis were found when the embryonal carcinoma cells were compared with the embryonic ectoderm (that portion of the ICM which remains after endoderm differentiation). Both synthesize at least one prominent 55,000-dalton protein that is not detected in embryonic cells at earlier stages of development. This protein can thus be used as a biochemical marker of ectoderm formation during embryonic development. The pattern of protein synthesis common to embryonal carcinoma cells and embryonic ectoderm is not shared by other cultured cell types.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

The transmission dynamic of different hepatitis B-infected individuals with the effect of hospitalization

We propose an epidemic model for the transmission of hepatitis B virus along with the classification of different infection phases and hospitalized class. We formulate the model and discuss its basic mathematical properties, e.g. existence, positivity, and biological feasibility. Exploiting the next generation matrix approach, we find the basic reproductive number of the model. We perform sensitivity analysis to illustrate the effect of various parameters on the transmission of the disease. We investigate stability of the equilibria of the model in terms of the basic reproduction number. Conditions for the stability of the proposed model are obtained using various approaches. Finally, we perform the numerical simulations to discuss sensitivity analysis and to support our analytical work.     

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.     

Lung proteome alterations in a mouse model for nonallergic asthma

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.     

Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing cholangitis

We investigated the changes in the hepatic proteome in murine models for toxic-induced fibrogenesis and sclerosing cholangitis. A comprehensive comparison of protein changes observed is made and the mechanistical basis of the expression changes is discussed. Hepatic fibrosis was induced by repetitive intraperitoneal CCl4 treatment of BALB/c mice or developed spontaneously in BALB/c-ATP-binding cassette, subfamily B, member 4 (Abcb4) knock out mice. Fibrosis was verified by a morphometric score and assessment of hydroxyproline content of liver tissue, respectively. The innovative difference in-gel electrophoresis (DIGE) technique was used to analyse protein expression levels of the mouse proteome. Results were confirmed by Western blotting and real-time RT-PCR. In CCl4-induced fibrosis 20 out of 40 and in BALB/c-Abcb4(-/-) mice 8 out of 28 differentially expressed proteins were identified utilizing DIGE. Only two proteins, selenium-binding protein (Sbp2) and carbonic anhydrase 3, have been unidirectionally expressed (i.e. down-regulated) in both models. Relevant differences in the pathogenesis of toxically induced liver fibrosis and sclerosing cholangitis exist. The only novel protein with regard to liver fibrosis depicting a unidirectional expression pattern in both animal models was Sbp2. An explicit protein function could not be clarified yet.     

Early dysregulation of the mitochondrial proteome in a mouse model of Alzheimer's disease

Mitochondrial structural and functional alterations appear to play to an important role in the pathogenesis of Alzheimer's disease (AD). In the present study, we used a quantitative comparative proteomic profiling approach to analyze changes in the mitochondrial proteome in AD. A triple transgenic mouse model of AD (3xTg-AD) which harbors mutations in three human transgenes, APP(Swe), PS1(M146V) and Tau(P301L), was used in these experiments. Quantitative differences in the mitochondrial proteome between the cerebral cortices of 6-month-old male 3xTg-AD and non-transgenic mice were determined by using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry. We identified 23 different proteins whose expression levels differed significantly between triple transgenic and non-transgenic mitochondria. Both down-regulated and up-regulated mitochondrial proteins were observed in transgenic AD cortices. Proteins which were dysregulated in 3xTg-AD cortices functioned in a wide variety of metabolic pathways, including the citric acid cycle, oxidative phosphorylation, pyruvate metabolism, glycolysis, oxidative stress, fatty acid oxidation, ketone body metabolism, ion transport, apoptosis, and mitochondrial protein synthesis. These alterations in the mitochondrial proteome of the cerebral cortices of triple transgenic AD mice occurred before the development of significant amyloid plaque and neurofibrillary tangles, indicating that mitochondrial dysregulation is an early event in AD.