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PubMed2004

Effect of low-power laser irradiation on protein synthesis and ultrastructure of human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Low-power lasers improve wound healing. Cell proliferation and protein secretion are important steps of this process. The aim of this study was to analyze both protein synthesis and ultrastructural morphology of human gingival fibroblasts irradiated by a low-power laser. STUDY DESIGN/MATERIALS AND METHODS: The cell line FMM1 was grown in nutritional deficit. Laser irradiation was carried out with a gallium-aluminum-arsenate (Ga-Al-As) diode laser (904 nm, 120 mW, energy density of 3 J/cm(2)). The protein synthesis analysis and ultrastructural morphology of control (non-irradiated) and irradiated cultures were obtained. RESULTS: There were changes in the structure of cytoplasm organelles of treated cells. The procollagen was not altered by the laser irradiation; however, there were a significant reduction of the amount of protein in the DMEM conditioned by irradiated cells. CONCLUSIONS: Low-power laser irradiation causes ultrastructural changes in cultured fibroblasts. We suggest that these alterations may lead to disturbances in the collagen metabolism.

Cells, Cultured↗
PubMed1984

Ultrastructure of trypan blue-induced ocular defects II. Cornea and mesenchyme.

Using scanning and transmission electron microscopy, the histological and cytological alterations induced in the developing eye by trypan blue were examined. Microphthalmic and anophthalmic eyes were analyzed from 16-day rat fetuses whose dams had received a teratogenic dose of trypan blue. Cell and tissue damage within cornea and mesenchyme were assessed. Corneas from the treated groups were undulating and uneven owing to abnormal lens placement and corneal cell injury. Extensive extracellular spacing, few cell-to-cell contacts, patchy glycocalyces, and abnormal surface morphology characterized the corneal cells of treated tissues. Mesenchyme of severely malformed eyes typically filled the eye field in regions normally occupied by retina and lens. As a tissue, mesenchyme lacked organization and those surface features normally present in component cells were altered. Mesenchymal cells were packed so closely that extracellular spaces were nearly obliterated. Microvilli, filopodia, and lamellipodia, cell processes observed in normal mesenchyme, were altered in both number and structure. The cytoplasm of mesenchymal cells was condensed and decreased in volume. These data suggested that 1) in this trypan blue ocular model, virtually all major tissue types were morphologically altered; 2) because healthy mesenchyme and cornea are required for normal development of lens, optic vesicle and ocular adnexa it follows that abnormal cornea and mesenchyme also will compromise these tissues; and 3) since necrotic reminants were not present, this trypan blue model probably causes developmental arrest. The most likely teratogenic pathway is presented with emphasis on the cell surface as a probable site of dye action.

Abnormalities, Drug-Induced↗
PubMed1999

Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta.

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell-shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts.

3T3 Cells↗
PubMed1993

The endoplasmic reticulum-sarcoplasmic reticulum connection. II. Postnatal differentiation of the sarcoplasmic reticulum in skeletal muscle fibers.

The postnatal differentiation of sarcoplasmic reticulum (SR) of rabbit skeletal muscles (the slow-twitch soleus and the fast-twitch adductor muscles) was monitored between Days 1 and 12 by following on Western blots the expression and accumulation of molecular markers specific not only for the muscle endomembrane system, i.e., calsequestrin (CS) and the ryanodine-sensitive Ca2+ release channel, but also for the endoplasmic reticulum (ER) at large, i.e., BiP, calnexin (CN) and calreticulin. Our results demonstrate that SR development, documented by the increase of the SR fractional volume, terminal cisternae proliferation, and reorientation of triads, is accompanied by the accumulation of the SR-specific proteins and also of CN, with no change of the other ER general markers. Moreover, the distribution of two of the markers, BiP and CS, was investigated by immunocytochemistry at both the light and the electron microscope level. At Day 1 CS was found to be concentrated both within the few recognizable triad terminal cisternae and within the lumen of numerous, apparently discrete cisternae and tubules, widely scattered throughout both the contractile and the subplasmalemmal areas of the cytoplasm. These structures remain evident until Day 12, when most triad junctions have acquired proper configuration, composition and orientation. BiP, on the other hand, appears widely distributed within the ER/SR of the fibers. From the early stages of postnatal development it does colocalize with the Ca2+ binding protein in the lumen of the CS-rich structures and appears also within the longitudinal SR and the conventional ER cisternae.

Animals↗
PubMed2000

Centrosome-specific perturbations during in vitro maturation of mouse oocytes exposed to cocaine.

Previous studies indicating that cocaine may perturb meiotic chromosome segregation in mammalian oocytes prompted an analysis of the effects of cocaine on mouse oocytes matured in vitro under defined exposure conditions. Cumulus-enclosed mouse oocytes were matured in vitro in the continuous presence of cocaine and assessed for meiotic cell cycle progression and centrosome-microtubule organization using a combination of cytogenetic and fluorescence microscopic techniques. Both of these approaches demonstrated that cocaine had little effect on meiotic cell cycle progression to metaphase of meiosis-2 except at the highest dose tested (1000 microg/ml) where progression from metaphase-1 to metaphase-2 was inhibited. Cytogenetic analyses further showed that bivalent segregation was moderately affected and the incidence of premature centromere separation was significantly decreased following cocaine treatment. Under conditions of cocaine exposure, striking changes in meiotic spindle structure and cytoplasmic centrosome organization were observed. A 36% reduction in spindle length was associated with a loss of nonacetylated microtubules and fragmentation of spindle pole centrosomes. Moreover, in oocytes exposed to cocaine during maturation, a doubling in cytoplasmic centrosome number was observed. These results are discussed with respect to the relative roles of chromosomes and centrosomes in establishing and maintaining functional microtubule organization during meiosis in oocytes.

Aneuploidy↗
PubMed1997

Ultrastructural and immunocytochemical studies of prolactin-secreting cells in adenohypophysis of the mink (Mustela vison).

This investigation aimed to identify, by the double immunogold procedure, the ultrastructural characteristics of prolactin (PRL) cells in the mink. Such cells showed a marked pleomorphism and had a close topographic relationship with growth hormone cells. A common morphological characteristic of PRL cells in all stages of mink development was the presence of round secretory granules, in contrast to changes in the ultrastructural characteristics of PRL cells with physiological state and photoperiod. Thus PRL cells in prepubertal, pubertal, and sexually inactive adult mink, killed under a short-day photoperiod, showed little development of the organelles but a significantly increased cytoplasmic electron density. In sexually active mink and in lactating females under long-day conditions, PRL cells had a highly developed cytoplasmic organelle structure consisting mainly of rough endoplasmic reticulum. The morphometric study demonstrated that the mean diameter of the secretory granules similarly varied in both sexes. Pubertal mink had PRL cells with smaller secretory granules (female 74.1 +/- 0.6 nm, male 80.4 +/- 1.7 nm), whereas adult mink killed under a long-day photoperiod presented PRL cells with larger secretory granules (female 194.5 +/- 2.2 nm, male 203.3 +/- 1.7 nm). The changes in the ultrastructural characteristics of PRL cells during the annual cycle suggest a photoperiodic influence upon these cells. In addition the heterogeneity in ultrastructural characteristics and storage characteristics of PRL in some adult mink may suggest a varying metabolic role for PRL under certain, as yet not fully characterized, conditions.

Aging↗
PubMed2002

Inducible system in human hepatoma cell lines for hepatitis C virus production.

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.

Carcinoma, Hepatocellular↗
PubMed2007

Somatic cell nuclear transfer (SCNT) in mammals: the cytoplast and its reprogramming activities.

It is now more than nine years since Dolly, the world's first somatic cell cloned mammal was born, and the success of somatic cell nuclear transfer (SCNT) is still disappointingly low. Only about 3-5% of reconstructed embryos develop to term, and it is also evident that even if some clones are born, they are not necessarily fully developed and healthy. Embryonic and neonatal abnormalities of cloned offspring are probably a result of incorrect or incomplete reprogramming of the transferred donor cell nuclei. Such an incomplete reprogramming reflects the extremely low efficiency of SCNT. The key role in the process of reprogramming has been attributed to the enucleated oocyte-cytoplast into which the somatic cell nucleus is transferred. In our chapter, we will discuss the methodological approaches used for the preparation of cytoplasts and their possible reprogramming activities.

Animals↗
PubMed1987

The electromotor system of the electric catfish (Malapterurus electricus): a fine-structural analysis.

The electromotor system of the electric catfish (Malapterurus electricus) consists of two large ganglion cells situated in the spinal cord, two single axons containing electric nerves and two large electric organs with several million electroplaque cells. The small, irregularly stacked electroplaque cells possess at their center a crater-like indentation from which a stalk like protrusion arises. Many synaptic contacts derived from a single axon collateral are carried on lobe-like protrusions at the terminal knob of this stalk. The electric nerve consists of a large myelinated axon (diameter: 25 micron) surrounded by many layers of connective tissue cells. The two ganglion cells (200 micron in diameter) are rich in elements of the rough endoplasmic reticulum, Golgi apparatus and lysosomal structures. The cytoplasm of the soma changes its appearance towards the voluminous axon hillock (50 micron in diameter) which these organelles do not enter. The cell soma is perforated in a tunnel-like manner by blood capillaries, axons and processes of glial cells. The cell soma and dendrites are covered with two types of synapse. One type forms mixed chemical and electrical (gap junctions) contacts with intermediate attachment plaques. The other type is only chemical in nature. This system may be useful in the study of an identified vertebrate giant neuron.

Animals↗
PubMed1990

Alpha-spectrin immunoanalog in Acanthamoeba cells.

A monospecific, affinity purified antibody was prepared against chicken erythrocyte alpha-spectrin. The antibody cross-reacted with only one high molecular weight polypeptide (235 kDa) from whole Acanthamoeba cells. The localization of alpha-spectrin-related antigen in Acanthamoeba cells was examined using immunofluorescence and postembedding cytochemical techniques. Three patterns of distribution of alpha-spectrin immunoanalog were distinguished: as submembranous layer, cytoplasmic aggregates and uniform dispersion through the cytoplasm. Immunoelectron microscopic studies showed that the colloidal gold label was located in the cytoplasm in the vicinity of the plasma membrane. The gold particles were also aggregated around unidentified cytoplasmic filamentous structures. The presence of spectrin-related protein in protozoan cells of Acanthamoeba is in accordance with previous assumptions of the widespread occurrence of spectrin-related proteins. The heterogenous distribution of the immunoanalog of alpha-spectrin protein in Acanthamoeba cells is discussed.

Acanthamoeba↗
PubMed1996

A monoclonal antibody against DNA topoisomerase II labels the axial granules of Pleurodeles lampbrush chromosomes.

By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodeles. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of approximately Mr 180000 and a structurally nonrelated cytoplasmic protein of Mr 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.

Animals↗
PubMed1991

Histopathogenesis of inflammatory linear verrucose epidermal naevus: histochemistry, immunohistochemistry and ultrastructure.

Skin lesions of three patients with inflammatory linear verrucose epidermal naevus (ILVEN) were examined. Histologically, orthokeratosis and parakeratosis were alternately seen in the acanthotic epidermis. By N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide staining, the horny cells in the parakeratotic epidermis showed a cytoplasmic SH pattern and a weak membranous SS pattern. The orthokeratotic epidermis revealed an increased involucrin expression, whereas the parakeratotic epidermis showed almost no involucrin expression. Ultrastructurally, in the parakeratotic epidermis, the living keratinocytes had prominent Golgi apparatuses and vesicles in the cytoplasm. In the intercellular spaces in the upper spinous layer through to the lower horny layer, an electron dense, homogeneous substance was deposited. The cytoplasm of the horny cells was filled with keratin filaments and contained remnants of nucleus and cytoplasmic membrane structures, and some lipid droplets. The marginal band formation was incomplete. Most of these ultrastructural abnormalities were not found in the orthokeratotic epidermis. There are both similarities and differences in histopathogenesis of the parakeratotic epidermis between ILVEN and psoriasis. A unique finding was the lack of involucrin expression in the ILVEN parakeratotic epidermis.

Adult↗
PubMed1976

Light- and electron-microscopic studies on the formation of "dark" variants of chromaffin cells in the rat adrenal medulla.

"Dark" cell formation has been studies in the adrenal medulla with combined light- and electron-microscopic methods. Correlation between light- and electron-microscopic appearance has been demonstrated. It has been pointed out that the formation of "dark" cell artefact can be prevented during immersion fixation and can be produced also during perfusion fixation by appropriate physical conditions. "Dark" cell formation was elicited by prefixation cooling and/or by increased tissular pressure. The nature of the underlying physicochemical change was interpreted as a uniformly distributed sol-gel transition of cytoplasmic macromolecules. The formation of cytoplasmic gel structures can be considered an endothermic process which involves volume increase (+ deltaV) - as may be inferred by their susceptibility to solation by high pressure and low temperature.

Adrenal Medulla↗
PubMed1991

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: light and electron microscopic study.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

Adult↗
PubMed1995

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber.

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocytochemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells; (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.

Animals↗
PubMed2006

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

Amino Acid Sequence↗