Specific interaction of lung surfactant protein A (SP-A) with rat alveolar macrophages

We have analyzed interaction of recombinant human surfactant protein A (SP-A) with isolated rat alveolar macrophages in the electron microscope. SP-A coated onto gold particles of different diameter is bound and internalized by macrophages. Binding and uptake occurs via coated membrane structures. SP-A gold particles are transported to secondary lysosomes. Binding and uptake is specific; i.e., excess of SP-A inhibits SP-A gold particle binding and uptake by 67% and depends on the presence of divalent cations. In experiments with ManBSA (5 x 10(-6) M) inhibition is 60%, but no inhibition occurs with GalBSA. The mannose-dependent interaction of SP-A particles with macrophages is not due to the mannose-specific receptor on the cell surface of macrophages as shown in experiments with macrophages exhibiting reduced mannose receptor activity. These cells show reduced binding and uptake of mannan gold particles (42% inhibition) but no reduction of SP-A gold particle binding and uptake. Furthermore, mannan gold particles do not compete with binding of SP-A gold particles.     

嫁接早期IAA在西葫芦／南瓜嫁接面处薄壁细胞和维管分子中的免疫 …

Immuno-gold localization of IAA in cells of the graft union in the explant internode graft of Cucurbita pepo/Cucurbita moschata were investigated with electron microscopy. In parenchyma cells near the graft union, the gold particles were mainly accumulated in nucleus, plastid and endoplasmic reticulum, while no gold particles was detected in Golgi body, mitochondrion, cell wall and vacuoles. In the differentiating xylem element, the gold particles were labeled in secondary wall and cytoplasm. In the sieve element gold particles were found in the sieve plate, sieve pore and cytoplasm. There was a dense label of the gold particles in the companion cell. The role of IAA in the differentiation of the vascular elements was discussed.     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles

The effects of small size (～2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the “free space” between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.     

Direct detection of immunogold reactions by real-time video microscopy

Summary Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

High-resolution immunocytochemical labeling of replicas with ultrasmall gold.

The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold probes for immunocytochemical labeling of replicas was examined. When HLA Class I in neutrophil membrane replicas was labeled with various sized immunogold particles as the secondary detection system, the apparent distribution density was inversely related to the size of the particles (1.4-nm > 5-nm >10-nm >15-nm). Indeed, the density of the apparent distribution of HLA Class I labeled with 1.4-nm gold particles was about sevenfold greater than when labeling was carried out with the 10-nm gold particles. Similar results were obtained with CD16, another neutrophil membrane protein. Silver enhancement was required to visualize the 1.4-nm gold particles, but this procedure did not adversely affect replica membranes. These results suggest that, when followed by silver enhancement, 1.4-nm gold particles are effective probes for achieving high-resolution immunocytochemical labeling of replicas.(J Histochem Cytochem 47:569-573, 1999)     

蚕豆（Vicia faba L．）叶肉细胞中ABA的胶体金免疫电镜定位

利用胶体金免疫电镜定位技术对蚕豆叶肉细胞中ＡＢＡ定位的研究表明,在以ＡＢＡ抗体处理的切片中,叶绿体有大量的金颗粒标记,细胞质和细胞核也有金颗粒标记,但液泡和细胞壁中没有金颗粒标记。免疫染色前用胰蛋白酶处理可显著增强金颗粒标记密度,而不用ＥＤＣ固定或以免疫前兔血清处理的切片中几乎没有金颗粒标记。本实验为蚕豆叶肉细胞中ＡＢＡ的分布提供了直接的证据并说明了该技术是研究ＡＢＡ定位的一种可靠的方法。     

Direct detection of immunogold reactions by real-time video microscopy.

Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

Movement of a karyophilic protein through the nuclear pores of oocytes

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.     

Electron-probe x-ray analysis of granules and particles found in aurosomes produced by colloidal gold

Aurosomes produced in the rabbit synovial membrane after intraarticular injection of colloidal gold were found to contain large spherical electron-dense granules and fine electron-dense particles. Electron-probe x-ray analysis demonstrated the presence of gold in the granules and iron in the particles. Sulphur and phosphorus were not detected in these aurosomes produced by colloidal gold. This is in contrast to the aurosomes produced by the soluble gold salt sodium aurothiomalate where besides gold, sulphur and phosphorus are easily detected.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microprojectile particle effect on stable transformation of black spruce via bombardment

Three types of microprojectile particles, 1.0-μm gold, 1.3-μm tungsten, and 1.6-μm gold, were studied for their effectiveness on genetic transformation of black spruce via bombardment with somatic embryos as the target tissue. Different particles resulted in different levels of transient expression of theGUS reporter gene; 1.0-μm gold particles produced the highest level of expression, and 1.6-μm gold particles produced the lowest level. Particle type also affected stable transformation; 1.0-μm gold particles had a 10-fold higher stable transformation efficiency than did 1.6-μm gold particles and a 2-fold higher efficiency than did 1.3-μm tungsten particles. This study indicates that microprojectile particle type and size are important in bombardment-mediated plant transformation.     

Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).     

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.     

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.     

嫁接早期IAA在西葫芦/南瓜嫁接面处薄壁细胞和维管分子中的免疫细胞学定位

采用免疫胶体金法对IAA在西葫芦／南瓜离体茎段嫁接早期发育时期在嫁接面处的分布进行了超微结构水平的定位。电镜观察表明在嫁接面处的薄壁细胞中IAA主要定位于细胞核、质体、内质网等细胞器上。在高尔基体、线粒体、细胞壁和液泡中,未发现胶体金颗粒的标记。在分化中的管状分子中,胶体金颗粒位于次生壁上和细胞质中。在筛分子分化过程中,IAA主要定位于筛板、筛孔和细胞质中。在伴胞中有较高的金颗粒密度。对于IAA在嫁接体维管分子分化过程中的作用进行了讨论。     

Controlled growth of colloidal gold particles and implications for labelling efficiency

Summary A new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm. The initial gold particle population, with an average diameter of 5.6±0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous. An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles. The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of -galactosidase on ultrathin cryosections of Escherichia coli cells. We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method. In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading. Therefore these gold probes are suitable for qualitative double labelling experiments. The quantitative aspect of immunolabelling is discussed.     

Increased H2O2, vascular endothelial growth factor and receptors in the retina of the BBZ/Wor diabetic rat

Hyperglycemia in diabetes induces increased levels of hydrogen peroxide (H2O2), a reactive oxygen species generated by reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nontoxic levels of H2O2 increase endothelial cell permeability. Using a model of non-insulin-dependent diabetes, the BBZ/Wor rat, we investigated retinal levels of H2O2, vascular endothelial growth factor (VEGF) and its receptors, VEGF-R1 and VEGF-R2 by transmission electron microscopy at sites of the blood-retinal barrier (BRB). H2O2 localization was done by the cerium NADH oxidase method, and extravasation of endogenous serum albumin was used to document disruption of the BRB. Higher levels of H2O2 were detected in blood vessels of diabetic (78.7 +/- 4.84%) as compared with vessels from nondiabetic rats (39.0 +/- 4.47%). VEGF immunoreactivity was statistically higher in the inner BRB (24.67 +/- 0.33 colloidal gold particles/63 microm2 vs. 21.52 +/- 0.43 colloidal gold particles/63 microm2, p = .0001) and outer BRB (42.56 +/- 0.45 colloidal gold particles/63 microm2 vs. 15.51 +/- 0.51 colloidal gold particles/63 microm2, p = .0001) of diabetic rats as compared with age matched nondiabetic control rats. VEGF-R1 immunoreactivity was significantly higher in diabetic retinas in both the inner BRB (21.66 +/- 0.75 colloidal gold particles/63 microm2 vs. 12.69 +/- 0.61 colloidal gold particles/63 microm2, p = .0001) and outer BRB (22.76 +/- 2.36 colloidal gold particles/63 microm2 vs. 8.53 +/- 2.67 colloidal gold particles/63 microm2, p = .0013). VEGF-R2 was statistically higher in the inner BRB (8.97 +/- 0.57 colloidal gold particles/63 microm2 versus 7.03 +/- 0.65 colloidal gold particles/63 microm2, p = .0419) but not in the outer BRB (29.42 +/- 1.25 colloidal gold particles/63 microm2 vs. 28.07 +/- 1.42 colloidal gold particles/63 microm2, p = .4889). H2O2 levels correlated with increased VEGF (correlation coefficient = 0.82, p = .001) in this model of nonproliferative diabetic retinopathy. These results support that hyperglycemia is one factor that induces retinal endothelial cells in vivo to increase H2O2 via NADH oxidase and stimulates increases in VEGF resulting in disruption of the BRB.     

Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments

Summary The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (<25 nm) gold particles into 40–50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (>25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but not 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.     

Colloidal gold particles as an incompressible atomic force microscope imaging standard for assessing the compressibility of biomolecules.

Colloidal gold particles have multiple uses as three-dimensional atomic force microscopy imaging standards because they are incompressible, monodisperse, and spherical. The spherical nature of the particles can be exploited to characterize scanning tip geometry. As uniform spheres, colloidal gold particles may be used to calibrate the vertical dimensions of atomic force microscopy at the nanometer level. The monodisperse and incompressible nature of the gold can be used to characterize the vertical dimensions of coadsorbed biomolecules. Simultaneous measurements of gold with tobacco mosaic virus show that, at the same applied vertical force, the tobacco mosaic virus is undamaged by blunt tips but is compressed or disintegrated under sharper scanning styli, suggesting that specimen degradation is partly a pressure-dependent effect.