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PubMed1982

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking-focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.

Actins↗
PubMedDate not supplied

A milestone in ribosomal crystallography: the construction of preliminary electron density maps at intermediate resolution.

Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution. These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy. To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate cluster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 A (1 A = 0.1 nm), respectively). For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles. The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes. These models also inspired modelling experiments according to results of functional studies, performed elsewhere, focusing on the progression of nascent proteins.

Base Sequence↗
PubMed1982

[The endometrium of the corpus uteri in scanning electron microscopy].

The studied material comprised samples of 31 cases of human endometrium. The material was divided into groups in relation to histological picture of the endometrium. It has been concluded that surface endometrial epithelium is characteristic of changes during menstrual cycles. We could observe cilliary and secretory cells in the epithelim. In pathological conditions of human endometrium the authors also observed the above mentioned two types of cells. The surface endometrial epithelium in pathological states is deprived of the features observed in luteal phase of the menstrual cycle.

Adult↗
PubMed2003

Adhesive interactions between CD34(+)-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy.

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34(+)-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34(+) progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF-alpha and hSCF for 5 days. Immature CD83(-) CD86(low) dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1). dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2). dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3). microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34(+)-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.

Antigens, CD34↗
PubMed1962

Cinemicrophotographic observations on granule lysis in polymorphonuclear leucocytes during phagocytosis.

Phagocytosis of yeast cell walls and of Bacillus megaterium by human, rabbit, and chicken polymorphonuclear leucocytes has been observed by phase contrast microscopy and recorded on motion picture film. In suitably thin preparations intracellular events could be visualized well. Lysis of cytoplasmic granules began early in the course of the ingestion process, rupture occurring only in granules adjacent to the microorganism being engulfed. Formation of a visible vacuole about the ingested particle frequently followed degranulation. Chicken polymorphonuclear leucocytes, with their large phase-dense granules, were particularly suitable subjects for observations on detailed morphologic aspects of granule lysis. Rupture took place rapidly (0.1 second or less); in place of the granule there appeared a clear zone, often with a small phase-dense round structure in its center. Also accompanying granule lysis was an increase in phase density of the adjacent surface of the microorganism. Over the course of the following few seconds the darkening on the organism faded, the dense small body disappeared from view, and the clear zone contracted towards the engulfed particle. The observations are discussed in relation to the hypothesis that fusion takes place between the granule membrane and the invaginated cell membrane overlying the ingested particle, with discharge of granule contents directly into the phagocytic vacuole.

Animals↗
PubMed1988

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro.

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4-5 hours at 37 degrees C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.

Acrosin↗
PubMed1985

Studies of otoconia in the developing chick by polarized light microscopy.

Using polarized light microscopy we were able to observe the mineralization patterns of embryonic and neonatal chick otoconia. We compared preparations of freshly dissected material spread under mineral oil to material that had been treated with various fixatives and dehydration agents. We found that the standard fixation agent, glutaraldehyde, and some immersion oils etched embryonic chick otoconia but that fixation with 70% acetone or 70% alcohol followed by dehydration to 100% acetone or 100% alcohol left the otoconia intact. The size and shape of freshly dissected chick otoconia observed with polarized light microscopy were similar to those of acetone-fixed, critical-point-dried material examined by SEM. Embryonic forms of otoconia were found to have a fluted pattern that was different in morphology from otoconia found in hatched chicks and adults. Embryonic chick otoconia did not exhibit the multifaceted surface morphology seen in embryonic rat otoconia. Comparisons of the same fields of otoconia under phase contrast microscopy and polarized light microscopy indicated that the freshly dissected otoconia of embryos exhibit little or no unmineralized (non-birefringent) material but that glutaraldehyde-fixed otoconia exhibited unmineralized areas where etching had occurred. Size frequency distributions of freshly dissected embryonic and mature otoconia in five ages of embryos and hatched chicks were consistent with a hypothesized developmental sequence of otoconia.

Animals↗
PubMed2006

Estimation of polymer-surface interfacial interaction strength by a contact AFM technique.

Atomic force microscopy (AFM) measurements were employed to assess polymer-surface interfacial interaction strength. The main feature of the measurement is the use of contact-mode AFM as a tool to scratch off the polymer monolayer adsorbed on the solid surface. Tapping-mode AFM was used to determine the depth of the scraped recess. Independent determination of the layer thickness obtained from optical phase interference microscopy (OPIM) confirmed the depth of the AFM scratch. The force required for the complete removal of the polymer layer with no apparent damage to the substrate surface was determined. Polypropylene (PP), low-density polyethylene (PE), and PP-grafted-maleic anhydride (PP-g-ma) were scraped off silane-treated glass slabs, and the strength of surface interaction of the polymer layer was determined. In all cases it was determined that the magnitude of surface interaction force is of the order of van der Waals (VDW) interactions. The interaction strength is influenced either by polymer ability to wet the surface (hydrophobic or hydrophilic interactions) or by hydrogen bonding between the polymer and the surface treatment.

Adsorption↗
PubMed1990

GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies.

GDA-J/F3 monoclonal antibody (MoAb) recognized an antigen in the fibrous sheath (FS) of human spermatozoa. This was based on: (i) intracellular localization of the antigen which was limited to the principal piece of the sperm tail; (ii) its absence from the cilia of trachea and nasal mucosa which lack FS; (iii) immunogold electron microscopy (IEM) which confirmed its ultrastructural localization to the FS. The antibody was used for the detection of abnormal germ cells in human semen. Nucleated cells other than spermatozoa (NCOS) obtained from oligozoospermic donors were screened with GDA-J/F3 MoAb using the indirect immunofluorescence test. The antibody which stains only the tails in normal cells, produced diffuse cytoplasmic immunofluorescence inside some spermatids. Using phase contrast microscopy, the tails in these spermatids were either present or undetectable. Electron microscopy studies of the NCOS showed the lack of the FS in those which had the tails (afibrous tail), or the presence of disorganized tails (tail dysgenesis) in the others. This antibody therefore provides a useful analytical tool for probing the FS as well as for the easy identification of certain abnormal germ cells in human semen.

Antibodies, Monoclonal↗
PubMed2004

Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide.

AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer. METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis. RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax. CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

Acetamides↗
PubMed1989

Statistical analysis of data on airborne asbestos levels collected in an EPA survey of public buildings.

Data from a recent EPA study of airborne asbestos levels in 49 buildings occupied by the General Services Administration (GSA) are statistically analyzed. The study focuses on differences between indoor and outdoor levels, and on differences among buildings with no asbestos-containing material (ACM) (6 buildings), buildings with ACM generally in good condition (6 buildings), and buildings containing damaged ACM (37 buildings). Seven indoor samples and a single outdoor sample were collected from most buildings and analyzed by TEM (transmission electron microscopy) using a direct preparation method. No statistically significant differences were detected in asbestos levels between indoors and outdoors or among the three categories of buildings. The average indoor concentration of asbestos was 0.00073 fibers/cc (f/cc) for all fibers and 0.00007 f/cc for fibers 5 microns or longer. The current OSHA occupational standard is 0.2 f/cc for fibers 5 microns or longer as measured by phase-contrast microscopy.

Air Pollutants, Occupational↗
PubMed1987

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.

Biodegradation, Environmental↗
PubMed2003

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

Animals↗
PubMed1990

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.

Animals↗
PubMed1995

Response of human gingival fibroblasts to prostaglandins.

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA1, PGA2, PGB1, PGB2, PGD2, PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGI2, 6-keto-PGF1 alpha, 9 alpha-11 alpha-methanoepoxy-PGF2 alpha, and thromboxane (TX) B2. PGA1 and PGD2 at 30 microM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 microM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 microM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.

Cell Division↗
PubMed2002

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin.

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Acrosin↗
PubMed2003

Image analysis of tendon helical superstructure using interference and polarized light microscopy.

Wave-like structures (WLS also known as crimp) have generally been reported to be planar structures. However, there is evidence that a helical superstructure, rather than a planar one, should be considered. Conditions dictated by supramolecular chemistry, molecular recognition and self-assembly favor the idea of a helical arrangement for collagen bundles in a supramolecular structure. The aim of this work is to provide additional data in support of a helical superstructure for collagen bundles in tendons. Cryosections of fixed flexor bovine tendons and sections of resin-embedded peeled rat tail were studied using polarized light, interference, and phase contrast microscopy. Image analysis was used to find appropriate mathematical descriptors for WLS. Interference colors due to the dispersion of birefringence allowed the detection of a gradual, intertwined twisted fiber organization in WLS, as the angle of the tendon axis was rotated relative to the polarizers. Helical movements of the images of the WLS bands were produced using animation methods. Interference microscopy revealed interference colors associated with different orientations and dry mass concentrations in the fibers, especially in tendon cross-sections, which also exhibited Maltese-cross birefringence images. Similar images were detected by interference microscopy, suggesting a spiral organization of fibers in the section plane. The helical orientation of the fibers was detected by focusing through different planes of sections. Based on a comparison of this superstructure with mesophases, the twisted grain boundary concept is considered to be the most appropriate for the classification of tendon WLS.

Animals↗