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At least 1,171 records · Page 65Linked to original sources
PubMed1990

Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy.

Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

Carcinoma, Transitional Cell↗
PubMed2006

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.

Animals↗
PubMed2005

Impairment of nuclear pores in bovine herpesvirus 1-infected MDBK cells.

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.

Animals↗
PubMed2005

Oligodendrocyte dysfunction after induction of experimental anterior optic nerve ischemia.

PURPOSE: The early response and survival of oligodendrocytes after axonal stroke and their potential contribution to neuronal survival in vivo have not been adequately addressed. The purpose of this study was to investigate the changes occurring in the retina and optic nerve (ON) in anterior ischemic optic neuropathy (AION), using a c-fos transgenic mouse model. METHODS: A new mouse model of AION (rodent AION) was developed to evaluate the in vivo stress response of oligodendrocytes and retinal ganglion cells (RGCs) in a transgenic mouse strain, using the immediate early stress-response gene c-fos, RT-QPCR technology, immunohistochemistry, and electron microscopy. Confocal microscopy was used with cell-specific antibodies to characterize the timing of cells responding to rAION. The TUNEL assay detected cells undergoing apoptosis. Ultrastructural changes were analyzed by electron microscopy. RESULTS: In rAION, oligodendrocytes rapidly respond in vivo to ischemic ON damage, with c-fos activation as an early detectable event. Early evidence of progressive oligodendrocyte stress, is followed by demyelination, wallerian degeneration of the ON, and oligodendrocyte and RGC death far from the primary lesion. CONCLUSIONS: After rAION induction oligodendrocytes, as well as RGCs, undergo progressive stress, with dysfunction and apoptosis. The findings lead to a proposal that progressive retrograde oligodendrocyte stress, away from the primary lesion, is an important factor after ischemic optic neuropathy. Postinduction demyelination must be addressed for effective neuroprotection of ischemic and hypoxic white matter.

Animals↗
PubMed2005

Optical sectioning microscopy.

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.

Animals↗
PubMed1996

Actin bundles in human hair follicles as revealed by confocal laser microscopy.

A confocal laser microscope was used to examine the distribution pattern of actin bundles in whole-mounts of human hair follicles stained with fluorescently labeled phalloidin. Actin bundles were found exclusively in the epithelial outer root sheath of the lower and middle portions of the follicle. In the growth stage, the lower follicle was characterized by well-developed actin bundles arranged circumferentially in the innermost and outermost cell layers of the outer root sheath. Actin bundles in the innermost cells were aligned end-to-end so that they formed complete circular bands surrounding the inner root sheath. In the outermost cells, actin bundles ran underneath the basal plasma membrane to which they attached at both ends. In contrast, in the quiescent stage, actin bundles in the lower follicle were disposed radially toward the follicle surface where they terminated perpendicular to the basal plasma membrane. In the middle follicle, circumferential actin bundles were found only in the intermediate layer of the outer root sheath throughout the hair cycle. Immunofluorescent anti-myosin and anti-alpha-actinin staining showed a striated pattern along actin bundles. Vinculin was localized at both ends of actin bundles, corresponding to the cell-to-cell or cell-to-substrate adherens junctions. Glycerinated follicles changed in shape on the addition of MgATP, suggesting a contraction of actin bundles. From these observations, we conclude that actin bundles in the hair follicle are comparable to stress fibers and that they serve as a tensile scaffold for the growth and integrity of the follicle.

Actinin↗
PubMed1999

High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.

BACKGROUND: Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. AIMS: To optimise sample requirement for SCGE and CLS. METHODS: Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. RESULTS AND CONCLUSION: Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.

Bone Marrow Cells↗
PubMed2006

In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy.

RATIONALE: Fibered confocal fluorescence microscopy (FCFM) is a new technique that produces microscopic imaging of a living tissue through a 1-mm fiberoptic probe that can be introduced into the working channel of the bronchoscope. OBJECTIVES: To analyze the microscopic autofluorescence structure of normal and pathologic bronchial mucosae using FCFM during bronchoscopy. METHODS: Bronchial FCFM and spectral analyses were performed at 488-nm excitation wavelength on two bronchial specimens ex vivo and in 29 individuals at high risk for lung cancer in vivo. Biopsies of in vivo FCFM-imaged areas were performed using autofluorescence bronchoscopy. RESULTS: Ex vivo and in vivo microscopic and spectral analyses showed that the FCFM signal mainly originates from the elastin component of the basement membrane zone. Five distinct reproducible microscopic patterns were recognized in the normal areas from the trachea down to the more distal respiratory bronchi. In areas of the proximal airways not previously biopsied, one of these patterns was found in 30 of 30 normal epithelia, whereas alterations of the autofluorescence microstructure were observed in 19 of 22 metaplastic or dysplastic samples, five of five carcinomas in situ, and two of two invasive lesions. Disorganization of the fibered network could be found on 9 of 27 preinvasive lesions, compatible with early disruptions of the basement membrane zone. FCFM alterations were also observed in a tracheobronchomegaly syndrome and in a sarcoidosis case. CONCLUSIONS: Endoscopic FCFM represents a minimally invasive method to study specific basement membrane alterations associated with premalignant bronchial lesions in vivo. The technique may also be useful to study the bronchial wall remodeling in nonmalignant chronic bronchial diseases.

Bronchi↗
PubMed2001

Epidermal nerve fiber density in sensory ganglionopathies: clinical and neurophysiologic correlations.

We assessed the involvement of somatic unmyelinated fibers in sensory ganglionopathies by skin biopsy and quantitative sensory testing (QST). Sixteen patients with ganglionopathy, 16 with axonal neuropathy, and 15 normal controls underwent skin biopsy at the proximal thigh and the distal leg. Intraepidermal nerve fibers (IENF) were immunostained by antiprotein gene product 9.5, and their linear density was quantified under light microscopy. Confocal microscopy studies with double staining of nerve fibers and basement membrane were also performed. Healthy subjects and neuropathy patients showed the typical proximodistal gradient of IENF density; in neuropathies, values were significantly lower at the distal site of the leg, confirming the length-dependent loss of cutaneous innervation. Conversely, ganglionopathy patients with hyperalgesic symptoms did not show any change of IENF density between the proximal thigh and the distal leg. The distinct pattern of epidermal denervation seen in sensory ganglionopathy reflected the degeneration of somatic unmyelinated fibers in a fashion that was not length-dependent, which was consistent with both clinical and neurophysiologic observations and supported the diagnosis.

Biopsy↗
PubMed1994

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1.

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC6 (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10-70 nm for MDCK cells and 20-90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.

Animals↗
PubMed2000

Water permeability measurement in living cells and complex tissues.

The identification of molecular water transporters and the generation of transgenic mice lacking water transporting proteins has created a need for accurate methods to measure water permeability. This review is focused on methodology to characterize water permeability in living cells and complex multicellular tissues. The utility of various parameters defining water transport is critically evaluated, including osmotic water permeability (P(f)), diffusional water permeability (P(d)), Arrhenius activation energies (E(a)), and solute reflection coefficients (sigma(p)). Measurements in cellular and complex tissues can be particularly challenging because of uncertainties in barrier geometry and surface area, heterogeneity in membrane transporting properties, and unstirred layer effects. Strategies to measure plasma membrane P(f) in cell layers are described involving light scattering, total internal reflection fluorescence microscopy, confocal microscopy, interferometry, spatial filtering microscopy, and volume-sensitive fluorescent indicators. Dye dilution and fluorescent indicator methods are reviewed for measurement of P(f) across cell and tissue barriers. Novel fluorescence and gravimetric methods are described to quantify microvascular and epithelial water permeabilities in intact organs, using as an example lungs from aquaporin knockout mice. Finally, new measurement strategies and applications are proposed, including high-throughput screening for identification of aquaporin inhibitors.

Animals↗
PubMed2000

Nuclear pore function viewed with atomic force microscopy.

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.

Adenosine Triphosphate↗
PubMed1992

Peptide-containing innervation of rat femoral lymphatic vessels.

The lymphatic vessels conduct lymph fluid, proteins, and potentially antigenic material from the interstitium back to the bloodstream via lymph nodes, where solids are removed by phagocytic cells and recirculating lymphocytes and immunoglobulins are added. Immunostaining for two general neuronal markers, protein gene product 9.5 (PGP 9.5), a cytoplasmic ubiquitin C-terminal hydrolase, and synaptophysin, a calcium-binding four-span integral synaptic vesicle membrane glycoprotein, disclosed an abundant innervation of the large femoral lymphatic vessels in rats. This confirms and extends earlier findings based on nonspecific intravital methylene blue and silver impregnation staining methods. Nerves containing neuropeptide Y, C-flanking peptide of neuropeptide Y, and tyrosine hydroxylase, markers of noradrenergic postganglionic sympathetic fibers, were frequent whereas immunoreactivity to vasoactive intestinal peptide, a neuropeptide present in many cholinergic parasympathetic nerve fibers, was sparse suggesting possible sympathetic and parasympathetic influences. Furthermore, calcitonin gene-related peptide- and substance P-containing fibers were also present in the walls of lymphatic vessels suggesting a possible sensory influence in the coordinated myogenic responses. By comparison to normal light microscopy, confocal microscopy was found useful to trace the perihilar penetration of blood and afferent lymphatic vessels in lymph nodes. PGP 9.5-immunoreactive fibers were found in and around lymph nodes suggesting that there is a neural regulation of lymphoid node function. Because of their distribution, peptide-containing nerves may participate in regulating the capacity of the lymphatic pumping activity, and may possibly exert paracrine effects on lymphocytes.

Acetylcholinesterase↗
PubMed1992

Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex.

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-streptavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

Adrenal Cortex↗
PubMed2006

Structural and molecular hair abnormalities in trichothiodystrophy.

We examined hair from 15 patients with trichothiodystrophy (TTD), a rare inherited disorder with brittle, cystine-deficient hair. They had a wide variety of phenotypes, from brittle hair only to severe intellectual impairment and developmental delay. Polarizing light microscopic examination showed alternating light and dark (tiger tail) bands under polarizing microscopy. Confocal microscopy captured structural features of breaks in intact TTD hairs. The autofluorescent appearance was regular and smooth in normal donors and markedly irregular in sections of TTD hairs possibly reflecting abnormalities in melanin distribution. Scanning electron microscopy revealed numerous surface irregularities. All TTD hair samples had reduced sulfur content. We observed an inverse correlation (R(val)=0.9) between sulfur content and percent of hairs with shaft abnormalities (trichoschisis, trichorrhexis nodosa, or ribbon/twist). There was no association between clinical disease severity and percent of abnormal hairs. Raman spectra of hairs from TTD patients and normal donors revealed a larger contribution of energetically less favored disulfide conformers in TTD hairs. Our data indicate that the brittleness of the TTD hair is dependent upon abnormalities at several levels of organization. These changes make TTD hairs excessively prone to breakage and weathering.

Adolescent↗
PubMed1996

Atomic (scanning) force microscopy in cardiovascular research.

The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The AFM images by using a sharp cantilever tip to sense the repulsive and attractive forces between the tip and the sample surface. The force of interaction is kept constant while raster scanning, resulting in images of the surface contours with molecular and, on hard inorganic surfaces, even atomic resolution. Movement of the cantilever in the Z plane is detected by a laser beam reflected off the cantilever to a photodiode system, a piezotube allows an X and Y raster, and a three-dimensional image results. Its capabilities include: (1) the three-dimensional imaging of membranes and biomolecules with molecular and submolecular resolution; (2) such imaging not only of dry specimens but of specimens in a physiologic solution, thereby allowing the investigation of dynamic processes in both viable biomolecules and living cells; (3) the sensing of charge and intermolecular interaction forces; (4) the chemical or biochemical modification of the cantilever tip, which allows the identification of specific structures and the measurement of specific interactions (e.g., a ligand-receptor interaction); (5) nanometer control of the position and force of the cantilever, which, in turn, allows the physical manipulation of biomolecules, the dissection of biological structures (e.g., the separation of one gap junctional hemichannel from its neighbor, thereby revealing normally inaccessible surfaces), the delivery of ligands, drugs, or other materials to specific locations, and the precise measurement of interacting forces at specific sites; and (6) the modification of the apparatus by adding complementary methodologies (e.g., magnetic resonance imaging, fluorescence microscopy, confocal microscopy, and perhaps electrophysiology). AFM, however, is only now being applied to biological research, many technical and methodologic problems exist, and a number of them are considered in this review. Little work has been done in cardiovascular research and the purpose of this review is to introduce this new and exciting approach to investigation.

Animals↗
PubMed2005

Characterization of cytolethal distending toxin of campylobacter species isolated from captive macaque monkeys.

An association between certain Campylobacter species and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examine Campylobacter species isolated from captive conventionally raised macaque monkeys for the presence of the cdtB gene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkey Campylobacter isolates and examinations by light microscopy, confocal microscopy, and flow cytometry. Although cdtB gene sequences were found in both Campylobacter jejuni and Campylobacter coli, the production of cytolethal distending toxin correlated positively (P < 0.0001) only with C. jejuni. We concluded that cytolethal distending toxin activity is a characteristic of C. jejuni. Our C. jejuni cdtB gene-specific PCR assay might be of assistance for differentiating toxigenic C. jejuni from C. coli in clinical laboratories.

Animals↗
PubMed1997

Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor.

Animals↗