Influence of RGD-containing oligopeptide-coated surface on bone formation in vitro and in vivo

New bone formation on an RGD-containing oligopeptide-coated surface in vitro and in vivo was investigated. The surface showed two-fold higher osteoblastic cell adhesion and differentiation in vitro, and revealed statistically significant in vivo bone formation compared with the control (P < 0.05).     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Thy-1 is an in vivo and in vitro marker of liver myofibroblasts

Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7 days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro. Jozsef Dudas and Tümen Mansuroglu contributed equally to this study. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 402, projects C6, D3, D4).     

生物体衰老与复制衰老--体内与体外研究

体外连续培养的细胞在有人数的细胞分裂后,更新换代合成ＤＮＡ及分裂的能力,最后导致增殖能力的丧失,但基本代谢过程仍能维持,这种现象称为复制衰老。本文讨论了复制衰老现象存在的普遍性,描述了衰老细胞伯特征,对复制衰老和生物体衰老之间的联系进行了重点分析。现有的研究虽然还不完全,但都提示复制衰老是生物体衰老在细胞水平上的反映,并充分肯定了复制衰老是一个较好的研究机体衰老的模型。     

Amphiphysin1 inhibits vitronectin-mediated cell adhesion,spreading, and migration in vitro

To investigate the regulatory mechanism of cell adhesion, we have searched for cellular inhibitory factors which prevent cell adhesion. The brain cytosol was found to inhibit the adhesion of various transformed cells to the substratum. An inhibitory 120-kDa protein was purified by sequential column chromatography. Peptide sequencing revealed that the protein is identical to amphiphysin1. GST-amphiphysin1 suppressed the attachment of HeLa cells to the plate when cells were cultured in the serum-containing medium. Vitronectin, a major cell-adhesive protein in serum and a ligand to alpha(v)beta3 integrin, was responsible for this cell attachment, and the vitronectin action was blocked by GST-amphiphysin1. GST-amphiphysin1 also inhibited the vitronectin-mediated spreading and migration of malignant melanoma cells. Furthermore, GST-amphiphysin1 bound directly to vitronectin. These findings point to the interesting possibility that amphiphysin1 could be a useful tool to inhibit cell-adhesive vitronectin.     

Different fermentation processes produced variants of an anti-CD52 monoclonal antibody that have divergent in vitro and in vivo characteristics

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.     

Cell adhesion and invasion inhibitory effect of an ovarian cancer targeting peptide selected via phage display in vivo

Organ-specific metastasis is of great importance since most of the cancer deaths are caused by spread of the primary cancer to distant sites. Therefore, targeted anti-metastases therapies are needed to prevent cancer cells from metastasizing to different organs. The phage clone pc3-1 displaying peptide WSGPGVWGASVK selected by phage display had been identified which have high binding efficiency and remarkable cell specificity to SK-OV-3 cells. In the present work, the effects of selected cell-binding phage and cognate peptide on the cell adhesion and invasion of targeted cells were investigated. Results showed that the adhesive ability of SK-OV-3 to extracellular matrix was inhibited by pc3-1 and peptide WSGPGVWGASVK, and pc3-1 blocked SK-OV-3 cells attachment more effective than the cognate peptide. The peptide WSGPGVWGASVK suppressed the cell number of SK-OV-3 that attached to HUVECs monolayer up to 24% and could block the spreading of the attaching cells. Forthermore, the cognate peptide could inhibit the invasion of SK-OV-3 significantly. The number of invaded SK-OV-3 cells and invaded SK-OV-3-activated HUVECs pretreated with peptide WSGPGVWGASVK was decreased by 24.3% and 36.6%, respectively. All these results suggested that peptide WSGPGVWGASVK might possess anti-metastasis against SK-OV-3 cells.     

Increase in fluidity in the membrane of MT3 breast cancer cells correlates with enhanced cell adhesion in vitro and increased lung metastasis in NOD/SCID mice

To study whether membrane fluidity of tumor cells have an influence on metastasis, MT3 breast cancer cells harvested during exponential growth and under confluent conditions were compared. Electron paramagnetic resonance (EPR) data revealed that, in comparison to growing cells, confluent cells have a significant higher fluidity in their membrane related to a higher relative portion of disordered domains and a reduced portion of the most ordered domains. Further, sialyl Lewis X and/or A ligand-mediated adhesion of these cells was 2-fold enhanced. Confocal laser scanning microscopy further demonstrated a higher motility of ligands in the membrane of confluent cells, together with an accumulation of these ligands in distinct areas. Both facts are suggested to be responsible for an enhanced cell adhesion observed. Finally, an increased number of large distinct metastatic foci was registered in lungs of mice after i.v. inoculation of confluent cells. The results indicate that domain organization and fluidity of the cell membrane affect tumor cell adhesion and can have in this way also an impact on the malignancy of breast cancer cells.     

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.     

Improving fluorescence-based assays for the in vitro analysis of cell adhesion and migration

Cell adhesion and cell migration are two primary cellular phenomena to be approached in vitro in order to allow for the effective dissection of the individual events and the unravelling of their underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also affords an efficient way of conducting larger basic and applied research screenings of the conditions affecting these processes and are potentially exploitable in the context of routine tests in the biological and medical fields. Therefore, there is a substantial interest in devicing more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescent cell tagging. In this article we describe three fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automatized for high-throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.     

Muscle satellite cell heterogeneity: in vitro and in vivo evidences for populations that fuse differently

During development, muscle growth results from the proliferation of satellite cells (SC) and their fusion with fibers. Several studies revealed heterogeneity of SC population notably based on the proliferation rate. Here, we examined the SC characteristics of turkey skeletal muscles in terms of proliferation and more specifically fusion, to define if the ability of these cells to fuse may represent a distinct characteristic between them and could be directly associated with their proliferation properties. Freshly extracted SC were plated in clonal condition and their proliferation rate was assessed 11 days later. To investigate the SC fusion behavior, in vitro and in vivo approaches were developed. Highly and slowly proliferative SC were initially labeled with a nuclear -galactosidase (-Gal) activity and co-cultured with differentiated primary cultures. After 5 days, distribution of -Gal positive (-Gal+) nuclei was examined. Also, the two labeled SC types were transplanted into different muscles in autologous model. One week later, number of -Gal+ nuclei per fiber and diameter of fibers displaying -Gal+ nuclei were determined. In vitro, we showed that SC from turkey skeletal muscle are present as a heterogeneous population in terms of proliferation. Examination of their fusion properties in vitro as well as in vivo revealed that highly proliferative SC exclusively exhibited fusion with differentiated myotubes or myofibers, whereas slowly proliferative SC mainly fused together. Collectively, these data demonstrate for the first time that SC with different proliferation rate also intrinsically differ in their fusion potential, suggesting distinct roles for these sub-populations in muscle growth.     

Critical role of lipid membranes in polarization and migration of cells: a biophysical view

Cell migration plays vital roles in many biologically relevant processes such as tissue morphogenesis and cancer metastasis, and it has fascinated biophysicists over the past several decades. However, despite an increasing number of studies highlighting the orchestration of proteins involved in different signaling pathways, the functional roles of lipid membranes have been essentially overlooked. Lipid membranes are generally considered to be a functionless two-dimensional matrix of proteins, although many proteins regulating cell migration gain functions only after they are recruited to the membrane surface and self-organize their functional domains. In this review, we summarize how the logistical recruitment and release of proteins to and from lipid membranes coordinates complex spatiotemporal molecular processes. As predicted from the classical framework of the Smoluchowski equation of diffusion, lipid/protein membranes serve as a 2D reaction hub that contributes to the effective and robust regulation of polarization and migration of cells involving several competing pathways.     

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

毛脚鵟细胞体外培养体系建立及三种组织来源细胞特性分析

用组织块培养法对毛脚鵟不同组织进行原代培养,获得了3种不同组织来源的细胞,并成功对细胞进行了冷冻保存和复苏。在传代培养过程中,对比分析了3种组织来源细胞的形态学、生长曲线、贴壁率、核型等生物学特性。形态学方面,3种来源细胞均为成纤维样细胞。对于3种组织来源细胞的贴壁能力分析显示,输卵管源细胞最强,肺源细胞和气管源细胞次之。3种不同组织来源细胞的倍增时间分别为(29.91±0.39)、(33.18±0.21)和(30.67±0.28)h,群体倍增次数分别为3.54±0.01、4.52±0.02和4.38±0.03。毛脚鵟细胞的染色体数目为2n=68,性染色体为典型的ZW型。本实验为今后毛脚鵟细胞利用、遗传信息的保存及生物学特性的深入研究提供实验材料和依据。