Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy

Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches.     

Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting.

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.     

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.     

药流绒毛中波形蛋白的免疫荧光和免疫电镜研究

目的：观察米非司酮对人孕早期绒毛组织中波形蛋白(Vimentin)表达的影响。方法：应用间接免疫荧光和胶体金标记免疫电镜技术,检测正常早孕人流电吸组和米非司酮组绒毛组织中波形蛋白的表达。结果：对照组绒毛中轴间质细胞、血管内皮细胞波形蛋白阳性表达,药流组荧光着色细胞则明显减少,表达强度显著降低;对照组绒毛间质细胞波形蛋白中间丝沿细胞的长轴呈良好的丝状排列,整齐、致密,胶体金颗粒成簇或散在分布于细胞核与细胞质之间除细胞器以外的细胞质所有部位,胞核呈阴性;药流组间质细胞中间丝明显辣少,出现断裂、排列紊乱,金颗粒分布明显稀疏。结论：米非司酮显著降低绒毛中波形蛋白表达水平并影响绒毛间质细胞的细胞骨架成分。     

Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy

Summary Stretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchiet al. (1988) J. Muscl. Res. Cell. Motility 9, 491–8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.     

Localization of fibronectin in atherosclerotic lesions by immunohistochemistry and immunoelectron microscopy]

This paper reports the results of a study on the distribution of fibronectin (FN), its form, character and source in atherosclerotic lesions, using immunohistochemistry (PAP method) and immunoelectron microscopic technique. The results showed that large amounts of FN were localized in fatty streaks, gelatinous lesions and early atherosclerotic plaques. The intima smooth muscle cells in atherosclerotic lesions synthesized more FN, and it is likely that FN represents a new marker of smooth muscle cell modulated from "contractile" to "synthetic" state. With the maturation of atherosclerotic plaque, FN did not fill the whole plaque but was concentrated only in the fibrous cap surface and basocentral part of the atheroma. We also proved that procollagen III peptide (PIIIP) distribution in atherosclerotic plaque was similar to that of FN.     

Morphology and immunoelectron microscopy of AIDS virus

Summary Lymphocytes infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus were examined by thin-section and immunoelectron microscopy, with horseradish peroxidase as an electron-dense marker. There was no evidence of an intracytoplasmic virion precursor, but viral antigen accumulated at the plasma membrane. Particles formed at the plasma membrane, and most virus was found in extracellular spaces. Mature virus had an envelope with fuzzy surface projections surrounding a centric core and an electron-dense eccentric nucleoid.With 4 Figures     

Localization of alpha-spectrin in chicken and monkey ventral horns by immunoelectron microscopy

Summary Localization of -spectrin in chicken and monkey ventral horns has been studied by immunoperoxidase techniques at the electron microscopic level. For this purpose, an antiserum specific for chicken -spectrin (240 kD subunit of spectrin) was prepared. The characteristics of the staining patterns of both chicken and monkey ventral horns were essentially identical. The reaction product for peroxidase was contained in the somata of large cells (presumably motor neurons), dendrites and axons. No specific staining was seen with either preimmune or blocked sera. The staining within the cell somata was primarily localized in cortical cytoplasm. Within dendrites and axons the immunocytochemical label was associated predominantly with the cortical cytoplasm and with microtubules. Staining was heavy over postsynaptic densities. Although presynaptic terminals showed weak staining as a whole, heavy staining was sometimes observed in areas adjacent to the presynaptic plasma membrane facing the postsynaptic density. These results indicate that spectrin distributes widely and functions in many biological activities in the nervous system.     

Localization of sodium-potassium adenosine triphosphatase in sheep myocardium by immunoelectron microscopy

Immunohistochemical localization of sodium and potassium dependent adenosine triphosphatase (Na,K-ATPase) employing polyclonal antibodies was carried out on sheep myocardium. The tissue was fixed with glutaraldehyde or a paraformaldehyde/lysine/periodate combination fixative and embedded in different embedding media. Ultrathin sections were labeled with rabbit anti-sheep Na,K-ATPase antiserum followed by sheep anti-rabbit immunoglobulin complexed with colloidal gold. Glycol methacrylate embedded tissue provided good structural preservation and showed specific immunohistochemical labeling. Analysis of the gold particle distribution showed a significantly higher number of Na,K-ATPase immunoreactive sites associated with cell membrane and T-tubules.     

Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy

To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or 10-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes.     

A simplified microwell pseudoreplica for the detection of viruses by electron microscopy and immunoelectron microscopy

Simplified procedures for immunoelectron microscopy (IEM) and electron microscopy (EM) are described. The procedures employ the principle of agar filtration and pseudoreplication. The modification consisted of the use of microwells for storage of gels with or without antiserum (for IEM or EM, respectively) and an array of containers in which pseudoreplication and negative staining were performed. The containers were prepared from 5 ml syringes from which the needle holding parts were cut. This device enabled simultaneous and rapid handling of specimens. With Sindbis virus as a model, our microwell pseudoreplica IEM (MW-PR-IEM) was compared to six other IEM techniques and was found to be the most rapid and sensitive technique. With the MW-PR-IEM technique, the specific minimal detection limit (detection of clumps) was 1.5 x 10(7) virus particles per ml, and the non-specific detection limit (detection of single virions) was 1.8 x 10(6) virus particles per ml.     

Direct demonstration of multiple VH allotopes on rabbit Ig molecules: allotope characteristics and Fab arm rotational flexibility revealed by immunoelectron microscopy

Immune complexes composed of rabbit IgG and Fab fragments of antibody specific for the VH framework allotypes were analyzed by molecular immunoelectron microscopy. In this manner, the number of allotypic epitopes ( allotopes ) and their approximate topological location could be determined. A monoclonal anti-allotype Fab was used to show that relatively fine details of allotope location and orientation are demonstrable with this technique. Each of the three VH allotypes (a1, a2 and a3) was found to express at least two spacially separated allotopes . The locations of the allotopes varied for each of the three allotypes. Some allotopes were observed near the VH-CH1 switch region while others were in close proximity to the complementarity-determining region or were at intermediate positions. In addition to the information concerning allotope topology, the variety of configurations resulting from the interaction of monoclonal antibody Fab with IgG suggest considerable rotational flexibility (twisting) of the Fab arm of IgG about its long axis.     

Bax, reactive oxygen, and cytochrome c release in neuronal apoptosis

Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.     

Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.     

Subcellular distribution of urokinase and urokinase receptor in human neutrophils determined by immunoelectron microscopy

A high-affinity receptor for urokinase-type plasminogen activator (uPAR) has been identified on the plasmamembrane of a number of different cell types, and has been shown to be important for plasminogen activation, cell adhesion, and possibly signal transduction. uPAR and uPA cosediment with secretory vesicles and specific granules by subcellular fractionation and translocate to the plasma membrane upon activation of neutrophils. Here the subcellular distribution of uPAR and uPA is studied by electron microscopy of neutrophils using immunogold double labeling for uPAR and uPA and a set of markers for well-defined subtypes of granules: matrix metalloproteinase type-9 (MMP-9) for gelatinase granules, lactoferrin (LF) for specific granules, and myeloperoxidase (MPO) and neutrophil elastase (NE) for primary granules. With this technique uPAR colocalizes with uPA in 71% of labeled granules. In granules containing uPAR the degree of coexpression with MMP-9, MPO and NE was 19, 66, and 74%, respectively. In granules labeled for uPA the corresponding overlap with MMP-9, MPO and NE was 24, 64, and 51%, respectively. Low levels of co-localization were found for uPAR and LF (7%) and for uPA and lactoferrin (5%). The results indicate that uPAR and uPA arepresent in gelatinase granules and primary granules, but rarely in specific granules. The demonstration of uPAR and uPA in primary granules is of particular interest, and may indicate that uPAR and uPA participate in the activation of latent hepatocyte growth factor of neutrophils.     

Thimerosal induces neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria

There is a worldwide increasing concern over the neurological risks of thimerosal (ethylmercury thiosalicylate) which is an organic mercury compound that is commonly used as an antimicrobial preservative. In this study, we show that thimerosal, at nanomolar concentrations, induces neuronal cell death through the mitochondrial pathway. Thimerosal, in a concentration- and time-dependent manner, decreased cell viability as assessed by calcein-ethidium staining and caused apoptosis detected by Hoechst 33258 dye. Thimerosal-induced apoptosis was associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. Although thimerosal did not affect cellular expression of Bax at the protein level, we observed translocation of Bax from cytosol to mitochondria. Finally, caspase-9 and caspase-3 were activated in the absence of caspase-8 activation. Our data suggest that thimerosal causes apoptosis in neuroblastoma cells by changing the mitochondrial microenvironment.     

Mitochondrial cytochrome c release in radiation-induced apoptosis of human peripheral T cells

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, using a microscopy-video system, mitochondrial membrane potential assay, annexin V, propidium iodide, and cytochrome c ELISA kit. After 5 Gy irradiation with 10 MV X-ray from a linear accelerator, the percentages of apoptotic T cells were estimated as approximately 5, 10, 20, 35, and 70%, at 0, 3, 6, 10, and 20 h after irradiation, respectively, as observed with the microscopy-video system. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, approximately half of the T cells showed dysfunction of mitochondrial membrane potential at 10 h after 5 Gy irradiation. With regard to annexin V and propidium iodide, approximately 40 and 5% of the human peripheral T cells showed positivity against annexin V and propidium iodide at that time, respectively. Mitochondrial cytochrome c release from the mitochondria to the cytosol was confirmed to start at 10 h and to reach a maximum at 20 h after 5 Gy of irradiation. These results demonstrated that mitochondrial cytochrome c release occurred following dysfunction of mitochondrial membrane potential in radiation-induced T cell apoptosis.     

Ultrastructural analysis of human T and TC mast cells identified by immunoelectron microscopy

Tryptase and chymase were localized in human mast cells by immunoelectron microscopy, enabling the T (tryptase positive, chymase negative) and TC (tryptase positive, chymase positive) types of mast cells to be identified and ultrastructurally characterized. A double immunogold staining procedure was performed on samples of human skin, small intestine, and lung with rabbit polyclonal IgG anti-chymase and mouse monoclonal IgG anti-tryptase primary antibodies and gold-conjugated secondary antibodies. Approximately 225 mast cells were examined in this fashion; comparable sections from 170 of these mast cells along with approximately 200 additional mast cells also were examined using techniques optimized for ultrastructural detail. Each secretory granule of TC mast cells contained both tryptase and chymase; secretory granules of T mast cells stained strongly positive for tryptase alone. Extremely small amounts of chymase appeared to be present in an occasional T mast cell granule. Staining for the neutral proteases was more intense over electron-dense regions of the granules, particularly noticeable over the characteristic discrete scrolls of T mast cells. T and TC mast cells each had large numbers of cytoplasmic granules, nuclei with peripherally condensed chromatin and low nuclear/cytoplasmic ratios, indicating maturity of both cell types. TC mast cell granules generally were more uniformly electron dense, larger and more numerous than T mast cell granules, which were more variable in shape. Compact solid-core scrolls, peripheral parallel lamellae and amorphous electron-dense material were found in granules of both cell types. Only TC mast cells had granules with grating and lattice substructures; only T mast cells had granules containing discrete scrolls. Less commonly, T mast cells were detected containing granules with a characteristic beaded or particulate ultrastructure. The ultrastructural features noted above were observed in T and TC mast cells regardless of the tissue in which they were examined and thereby permit T and TC mast cells to be distinguished by ultrastructure alone.     

早期激素性股骨头坏死过程中细胞色素c的作用

BACKGROUND: Studies have shown that apoptosis is closely related to the pathogenesis of steroid-induced femoral head necrosis. The release of cytochrome c plays a very important role in the process of apoptosis. OBJECTIVE: To observe the effects of cytochrome c on early steroid-induced femoral head necrosis. METHODS: Twenty-four healthy adult male New Zealand rabbits at 5 months old were randomly divided into model group and control group (n=12 per group). Models of early steroid-induced femoral head necrosis were established by intragluteal injection of hormone combined with ear vein injection of horse serum. In the control group, rabbits were given ear vein injection of the same amount of physiological saline. At 2, 4 and 6 weeks after model establishment, histopathological changes of bilateral femoral head were observed by optical microscope, and the ratio of empty lacuna was calculated. Apoptosis of osteocytes was determined by TUNEL assay, and apoptotic index was calculated. Immunohistochemical method was used to determine cytochrome c and to calculate cytochrome c-positive expression rate. RESULTS AND CONCLUSION: (1) The ratio of empty lacuna and apoptotic index: The model of early steroid-induced femoral head necrosis was successfully established in the experiment. Compared with the control group, ratio of empty lacuna, apoptotic index and expression rate of cytochrome c in osteocytes were significantly higher in the model group (P < 0.05 or P < 0.01). (2) Correlation analysis: Ratio of empty lacuna was significantly positively associated with apoptotic index at various time points in the model group (r=0.856, P < 0.01). Expression rate of cytochrome c was significantly positively associated with apoptotic index at various time points in the model group (r=0.824, P < 0.01). (3) These findings confirm that cytochrome c-involved apoptosis of osteocytes may play an important role in steroid-induced femoral head necrosis. Expression rate of cytochrome c in osteocytes is remarkably positively associated with the occurrence of steroid-induced femoral head necrosis in rabbits. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程