Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.     

Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes

Tissue-inherent factors such as cell-cell and cell-extracellular matrix interactions are regarded to exert a potentially large impact on adipogenesis as well as on secretory functions of adipose tissue. However, an appropriate 3-D adipogenesis model useful for addressing such interactions is still lacking. In this study, using tissue-engineering techniques, we demonstrate for the first time the development of coherent fat pads consisting of unilocular signet-ring cells in vitro. The constructs were generated by differentiating 3T3-L1 preadipocytes on 3-D polymeric scaffolds for either 9, 21, or 35 days in vitro. Only long-term culture yielded uniform tissues histologically comparable to native fat. Light and scanning electron microscopy provided direct evidence of 3-D tissue coherence and cell-cell contact in a tissue context, which was in strong contrast to conventional 2-D monolayer culture. Further differences between the two culture systems included enhanced secretion of leptin in 3-D tissue culture and differences in laminin expression (mRNA and protein level). Increase of triglyceride content over culture time and mRNA expression of other adipocyte genes, such as PPARgamma and Glut-4, were found to be similar. Implantation of long-term differentiated tissue constructs in nude mice resulted in further development and maintenance of fat pads. The presented model system is suggested to contribute to a better understanding of adipose tissue development and function facilitating studies on tissue-inherent interactions in vitro and in vivo.     

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.     

Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut

Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.     

Adipocyte differentiation of multipotent cells established from human adipose tissue

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist > beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.     

树莓的组织培养及快速繁殖

以树莓的茎尖为外植体进行组织培养,筛选出各培养阶段适宜的培养基分别为:(1)丛生芽诱导:MS 6-BA 1.0 mg/L;(2)继代培养:MS 6-BA 0.5~1.0 mg/L;(3)生根培养:1/2MS NAA 0.2mg/L或1/2MS IBA0.2 mg/L。     

桃叶杜鹃组织培养技术研究

选用桃叶杜鹃试管苗的芽苗、叶片进行离体培养,通过正交试验设计并结合方差分析、多重比较从中选出了不同中间繁殖体的最佳培养基配方.试验结果表明:改良MS和6-BA及KT不适合桃叶杜鹃的组织培养,相对较低浓度的TDZ却能诱导植株分化,建立起桃叶杜鹃的离体培养体系,达到保护名贵杜鹃的种质资源,加速繁殖的目的.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

A new approach to tissue engineering of vascularized skeletal muscle

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo-tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous-loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantaion of carboxy-fluorescine diacetate-succinimidyl ester (CFDA)-labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin-matrices without AV-loops. Intra-arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA-fluorescence, still showing positive MyoD staining. RT-PCR revealed gene expression of MEF-2 and desmin after 4 weeks on the AVloop side, whereas expression analysis of myogenin and MHC(embryo) was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre-vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle-like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.