Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

Egonol龙胆三糖苷及Egonol衍生物促雌二醇生成活性研究

对Egonol龙胆三糖苷及以Egonol衍生物对雌二醇生成活性及其相关机制进行了研究。发现Egonol龙胆三糖苷促雌二醇最高生成率在MCF-7、HepG2、ROS1728中分别为157% 、182.4%、226.8%（以空白组200μg/ml睾酮转换成E2值作为100%生成率）。活性的强弱可能与芳香化酶的组织特异性表达情况一致，说明Egonol龙胆三糖苷促雌二醇活性可能与芳香化酶有关。芳香化酶的组织特异性表达与特异性启动子有关系，Egonol龙胆三糖苷在各组织中皆有促雌二醇活性，说明该化合物不是通过调节该酶的基因表达而起作用。 在探究Egonol龙胆三糖苷及其衍生物是否介导cAMP-PKA途径从而影响芳香化酶的表达中，发现该系列化合物在HEK-293T细胞中对cAMP的影响非常弱小。在人HepG2细胞中显示了极强的提高cAMP的作用。而化合物对cAMP的作用与其促雌二醇活性强弱不呈正相关关系，对c AMP-PKA途径的激活可能与胞内雌激素有关。 Egonol龙胆三糖苷及其衍生物对HepG2细胞增殖影响显示，该系列化合物同雌二醇一样有相似的较弱促HepG2细胞增殖作用。而且存在一定剂量依赖性。在瞬时转染有ERE（雌激素作用元件）的HepG2中，Egonol龙胆三糖苷及其衍生物也显示了类似于雌二醇与ERE结合的作用，进一步提示Egonol龙胆三糖苷及其衍生物在HepG2细胞中具备雌激素样作用。 为研究Egonol龙胆三糖苷及其衍生物是否可能直接提高芳香化酶的活性，我们计划将芳香化酶从芳香化酶阳性细胞中克隆后表达到芳香化酶阴性的细胞中。在MCF-7细胞中以Oligo dT为引物合成的cDNA模板，和在ROS1728细胞中以Oligo dT及大鼠引物F链为引物合成的cDNA模板能成功扩增出与芳香化酶全长编码序列大小一致的片段。 Egonol衍生物在HepG2、ROS1728细胞中促雌二醇活性的实验表明，Egonol苯环上引入其它基团可以提高Egonol的活性。 从雌激素经典的基因组效应和非基因组效应两方面对雌激素信号转导研究进展进行了简单的综述。 The promoting effects of egonol gentiotrioside and egonol derivatives on the synthesis of estrogen E2 were studied. In vitro test, egonol gentiotrioside promoted the synthesis of estrogen E2 in MCF-7, HepG2,ROS1728 cell lines with mean yields of estrogen E2 57%,82.4% and 126.8%, higher than those of blank control at a concentration of 100 mg/ml. The difference of estrogen E2 synthesis promoting effects among the cell lines suggested tissue specificity. It is in accordance with tissue specific character of aromatase expression. The evidence implied that effect of egonol gentiotrioside on promoting the synthesis of estrogen E2 was related to the aromatase. Different expression levels of aromatase in different tissues are attributed to their specific promoters, but egonol gentiotrioside can promote the synthesis of estrogen E2, in many tissues,so the fact is controversary to the estimation that this compound regulates the aromatase on gene level. In order to investigate whether egonol gentiotrioside and its synthetic derivatives regulates aromatase activity through the cAMP-PKA signal pathway,we transfected the p CRE-Luc luciferase reporter gene into the HEK-293T cells and HepG2 cells. These compounds had weak activity in promoting the cAMP activity in HEK-293T cells but strong in HepG2 cells.The compounds’effect of promoting the cAMP may be related to their estrogenic activity in cells. The modified HepG2 cell proliferation assay was used to evaluate the estrogenic activity of egonol gentiotrioside and its derivatives. The weak estrogenic activity of egonol gentiotrioside and its derivatives at various concentrations expressed as proliferative effect relative to that of blank control was examined. We transfected the pERE-Luc luciferase reporter gene into the HepG2 cells. These compounds possessed significant activity on estrogen response element compared with the one treated with 10 n M estrogen E2. This evidence indicated that the estrogenic activity of egonol gentiotrioside and its derivatives. In order to investigate whether the egonol gentiotrioside and its derivatives can upregulate the activity of aromatase directly, The full-length of P450 aromatase cDNA encoding aromatase were amplified by using primer Oligo dT in MCF-7,and specific primer in ROS1728,respectively. The structure-activity relationship of Egonol in promoting the synthesis of E2 in HepG2 and ROS1728 cells indicated that introduction of some group on the basic sketon of egonol could improve the effect. The progress in research of signal pathway of estrogen in recent years was summarized.

The effect of nocodazole on the transfection efficiency of lipid-bilayer coated gold nanoparticles

Nonviral vectors are safer than viral systems for gene therapy applications. However, the limited efficacy always prevents their being widely used in clinical practice. Aside from searching new gene nonviral vectors, many researchers focus on finding out new substances to improve the transfection efficiency of existent vectors. In this work, we found a transfection enhancer, nocodazole (NCZ), for dimethyldioctadecylammonium (DODAB, a cationic lipid) bilayer coated gold nanoparticles (AuNPs) mediated gene delivery. It was found that NCZ produces 3-fold transfection enhancement to HEK 293T cells assessed by flow cytometry (FCM). The result was further confirmed by luciferase assay, in which NCZ induced more than 5 times improvement in transfection efficiency after 48 h of transfection. The results from the inductively coupled plasma mass spectrometry (ICP-MS) and FCM showed that NCZ did not affect the internalization of DODAB-AuNPs/DNA complexes. The trafficking of the complexes by transmission electron microscopy (TEM) indicated that the interrupted transportation of the complexes to the lysosomes contributed greatly to the transfection enhancement.

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Poly(L-lysine)-graft-chitosan copolymers: Synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by H-1 NMR, C-13 NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 similar to 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.

Telomerase Activity and Microgravity-Dependent Apoptosis in Human Leukemia HL-60 Cells

Mammalian cells subjected to conditions of spaceflight and the microgravity environment ofspace; manifest a number of alterations in structure and function. Among the most notable changes incells flown on the Space Shuttle are reduced growth activation and decline in growth rate in the totalpopulation. Other changes include chromosomal aberrations, inhibited locomotion, alteredcytokine production, changes in PKC distribution, and increased apoptos.

Two-Phase Flow In Fuel Cells In Short-Term Microgravity Condition

A visual observation of liquid-gas two-phase flow in anode channels of a direct methanol proton exchange membrane fuel cells in microgravity has been carried out in a drop tower. The anode flow bed consisted of 2 manifolds and 11 parallel straight channels. The length, width and depth of single channel with rectangular cross section was 48.0 mm, 2.5 mm and 2.0 mm, respectively. The experimental results indicated that the size of bubbles in microgravity condition is bigger than that in normal gravity. The longer the time, the bigger the bubbles. The velocity of bubbles rising is slower than that in normal gravity because buoyancy lift is very weak in microgravity. The flow pattern in anode channels could change from bubbly flow in normal gravity to slug flow in microgravity. The gas slugs blocked supply of reactants from channels to anode catalyst layer through gas diffusion layer. When the weakened mass transfer causes concentration polarization, the output performance of fuel cells declines.

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.

Two-phase Flow in Fuel Cells in Short-term Microgravity Condition

A visual observation of liquid-gas two-phase flow in anode channels of a direct methanol proton exchange membrane fuel cells in microgravity has been carried out in a drop tower. The anode flow bed consisted of 2 manifolds and 11 parallel straight channels. The length, width and depth of single channel with rectangular cross section was 48.0 mm, 2.5 mm and 2.0 mm, respectively. The experimental results indicated that the size of bubbles in microgravity condition is bigger than that in normal gravity. The longer the time, the bigger the bubbles. The velocity of bubbles rising is slower than that in normal gravity because buoyancy lift is very weak in microgravity. The flow pattern in anode channels could change from bubbly flow in normal gravity to slug flow in microgravity. The gas slugs blocked supply of reactants from channels to anode catalyst layer through gas diffusion layer. When the weakened mass transfer causes concentration polarization, the output performance of fuel cells declines.

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane-anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm(-1), which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm(-1) band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright (c) 2005 John Wiley T Sons, Ltd.

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis. (c) 2006 Optical Society of America.