The effects of naproxen sodium on specific parameters of L-strain cells over guided bone regeneration barrier materials.

There is strong evidence that nonsteroidal antiinflammatory drugs (NSAID) may exert a significant antiproliferative effect. This study evaluated the influence of NSAID on specific parameters of fibroblastic cells, in vitro, over two-guided bone regeneration (GBR) barrier materials. Fibroblast cells were cultured on bioabsorbable membrane made of collagen (Bio-Gide(R)- BG) and the most common nonresorbable membrane which is made of expanded polytetrafluoroethylene (ePTFE, Gore-Tex(R)- GT). Naproxen sodium (10 mM) was used as an analgesic drug. The fibroblast cells were cultured in vitro for 24 h and examined by scanning electron microscopy (SEM). Cells were cultured in the presence of (3)H-thymidine to study cell proliferation. And also cell numbers and viabilities were measured. The difference between the means for each group were analyzed for statistical significance by Kruskal-Wallis one-way ANOVA followed by post hoc comparisons using the Dunn statistical method. Of all the six groups, the control group stimulated DNA synthesis more than the others. With respect to cell numbers, there was statistically significant difference between the control group and naproxen planted BG membrane group. The interpretation of our SEM images is that these two barriers and naproxen seem to have had the least effect on cellular morphology. These data suggest naproxen have an inhibitory effect on stimulation of DNA synthesis, cell numbers and viabilities. And also lacking adherence of cells to the membranes may be due to the physical properties of the materials.

The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells.

Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus-cell interactions. The properties of uninfected and HSV-1-infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV-1-infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de-envelopments and re-envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV-1-induced cytoskeletal changes.

In vitro cellular accumulation of gadolinium incorporated into chitosan nanoparticles designed for neutron-capture therapy of cancer.

The accumulation of gadolinium loaded as gadopentetic acid (Gd-DTPA) in chitosan nanoparticles (Gd-nanoCPs), which were designed for gadolinium neutron-capture therapy (Gd-NCT) for cancer, was evaluated in vitro in cultured cells. Using L929 fibroblast cells, the Gd accumulation for 12 h at 37 degrees C was investigated at Gd concentrations lower than 40 ppm. The accumulation leveled above 20 ppm and reached 18.0+/-2.7 (mean+/-S.D.) microg Gd/10(6) cells at 40 ppm. Furthermore, the corresponding accumulations in B16F10 melanoma cells and SCC-VII squamous cell carcinoma, which were used in the previous Gd-NCT trials in vivo, were 27.1+/-2.9 and 59.8+/-9.8 microg Gd/10(6) cells, respectively, hence explaining the superior growth-suppression in the in vivo trials using SCC-VII cells. The accumulation of Gd-nanoCPs in these cells was 100-200 times higher in comparison to dimeglumine gadopentetate aqueous solution (Magnevist), a magnetic resonance imaging contrast agent. The endocytic uptake of Gd-nanoCPs, strongly holding Gd-DTPA, was suggested from transmission electron microscopy and comparative studies at 4 degrees C and with the solution system. These findings indicated that Gd-nanoCPs had a high affinity to the cells, probably contributing to the long retention of Gd in tumor tissue and leading to the significant suppression of tumor growth in the in vivo studies that were previously reported.

Characteristic biological effects of itraconazole on L929 fibroblasts and their cell membrane.

Itraconazole (ITCZ), a triazole antifungal agent, was studied for its effects on the morphology and function of L929 fibroblasts. L929 fibroblasts were cultured for 20 h with ITCZ or one of several other triazoles (fluconazole, ketoconazole, and hydroxy-itraconazole [ITCZ-OH]) at the concentration of 0.5 microg/ml. Among these agents, only ITCZ and its metabolite ITCZ-OH markedly elongated the cells bidirectionally. Scanning electron microscopy studies showed that the surface of the elongated cells was smoother than that of the untreated cells. The viability of L929 cells cultured with 0.5 microg/ml of ITCZ for 20 h was not lowered. However, after treatment with 0.0375% sodium deoxycholate (DOC) solution, the viability of the cells treated with ITCZ, as evaluated by the 3-(4,5-dimethyl-2thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay or the release of lactic dehydrogenase from cytoplasm, was decreased. When L929 cells were cultured in the presence of a combination of ITCZ and vincristine, their growth was synergistically inhibited. This synergism was also observed when ITCZ was replaced by ITCZ-OH, but not by the other azoles. These findings suggest that the exposure of L929 fibroblasts to low ITCZ concentrations affects the physiological nature of their cell membrane.

Neurite formation induced in neuroblastoma cells and genetically altered non-neuronal cells.

Ultrastructural changes associated with neurite formation were examined in suitably cultured neuroblastoma cells (Neuro-2a) and drebrin (developmentally regulated brain protein) gene-transfected fibroblasts (L cells) in culture. Both neuroblastoma cells and fibroblasts initially were flattened and epitheloid, with many microspikes or microvilli diffusely distributed over their surfaces. Intracellular organelles were abundant and diffusely arranged. In the transformed state, both cell types were round to oval with long processes where microspikes were concentrated. A constant arrangement of surface and intracellular structures was apparent, though drebrin gene-transfected fibroblasts retained some of their original characteristics. Neurite formation programmed by genes may be initiated by environmental factors in neuroblastoma cells. Neurite-like processes in fibroblasts may be formed due to changes in microfilaments resulting from transfection of the drebrin (actin-binding protein) gene.

Scanning electron microscopy of C-banding.

The mechanism of C-banding was analyzed on the basis of the structural changes of the 30 nm chromatin fibre using scanning electron microscopy (SEM). SEM of non-banded metaphase spreads of L-cells revealed chromosomes consisting of 30 nm chromatin fibre loops along the entire length. No marked difference in both the dimension and appearance of such looped structures was discernible between the centromeric region and the rest of the chromosome. In contrast, C-banded chromosomes exhibited a conspicuous alteration of the fibre conformation in the centromeric region. The looped, fibrous structures were almost completely lost from this region, while the non-centromeric region still exhibited fibrous structures with slightly different appearances compared with those observed in the control chromosomes. On the other hand, results obtained using fluorescence microscopy showed that more DNA retained in the centromeric region than in the non-centromeric region. Since the analytical experiments exhibited that the characteristic collapsed state of the centromeric region occurred only with the alkali treatment but neither with the 2 x SSC nor acid treatments, the centromeric heterochromatin seemed to contain some specific protein which should be sensitive to alkali. The structurally collapsed but subsequently compact centromeric region may become more, or still, resistant to the DNA extraction due to the 2 x SSC treatment and the centromeric chromatin thus retained may be visualized as the C-band.

The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus.

FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i.e. in microtubule depolymerizing conditions). Exposure of cells to 1 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination of the chemotherapeutic agent 5-fluorouracil with an inhibitor of its catabolism results in increased micronucleus induction.

The rate limiting step in 5-fluorouracil catabolism is catalyzed by the enzyme dihydropyrimidine dehydrogenase. Since degradation of 5-fluorouracil decreases its efficacy in chemotherapy, the inhibition of its catabolism is a promising tool. We investigated the formation of micronuclei in vitro in mouse L5178Y cells. 5-fluorouracil induced an increase in micronucleus frequency, which could significantly be enhanced by the concurrent application of 2,6-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase. The 5-fluorouracil concentration necessary to reach maximal genotoxic effects could be reduced to half in the presence of inhibitor 2,6-Dihydroxypyridine alone and the naturally occurring enzyme substrate uracil did not induce micronucleus formation. Combined application of the chemotherapeutic agent 5-fluorouracil and an inhibitor of its could reduce side-effects by lowering the effective dose of the active drug. With this study we provide further support for the usefulness of this concept.

Characterization of cadherin-4 and cadherin-5 reveals new aspects of cadherins.

Several properties of cadherin-4 and cadherin-5 were characterized by using the cDNA transfection approach. The proteins of both cadherins had a relative molecular mass of about 130 kDa and were present at the cell periphery, especially at cell-cell contact sites. These cadherins were easily digested with trypsin, and Ca2+ protected cadherin-4, but not cadherin-5, from the digestion. In immunoprecipitation, cadherin-4 co-precipitated with two major proteins of 105 kDa and 95 kDa, respectively. The 105 kDa and the 95 kDa proteins are likely to correspond to alpha- and beta-catenins. Cadherin-5 co-precipitated with only one major protein of 95 kDa, but seems to associate with the 105 kDa protein. On the other hand, plakoglobin or gamma-catenin did not co-precipitate well with either cadherin-4 or cadherin-5 in immunoprecipitation, but plakoglobin also appears to associated weakly with these cadherins. Cadherin-4 transfectants aggregated within 30 minutes in a cell aggregation assay, but cadherin-5 transfectants did not aggregate under the same conditions. Furthermore, the transfectants of chimeric cadherin-4 with cadherin-5 cytoplasmic domain showed cell aggregation activity comparable to that of wild-type cadherin-4 transfectants, whereas the transfectants of chimeric cadherin-5 with cadherin-4 cytoplasmic domain did not show appreciable cell aggregation, suggesting that the extracellular domains of cadherins, in conjunction with their cytoplasmic domains, play an important role in cell aggregation activity. These results show that cadherin-4 is very similar to the classical cadherins, whereas cadherin-5 is functionally as well as structurally distinct from classical cadherins.

Localization of dynamin: widespread distribution in mature neurons and association with membranous organelles.

Tissue distribution and intracellular localization of dynamin by immunoblotting and immunocytochemistry is investigated in this study. Dynamin was widely expressed in all the neurons we examined, and was especially abundant in the central nervous system after maturation, although its expression presented regional heterogeneity. Dynamin was present most abundantly in cerebellar Purkinje cells and hippocampal pyramidal cells, and to a lesser extent in motor neurons and peripheral nerves. However, dynamin was nearly absent in cells such as anterior pituitary cells and adrenal medullary cells which secrete mainly dense cored vesicles. Dynamin was localized not only in cell bodies, axons, and synapses but also in dendrites. Subcellular fractionation indicated that dynamin existed in the membrane fraction as well as in the soluble fraction. In ligated peripheral nerves, dynamin colocalized with tubulovesicular membranous organelles transported mainly anterogradely. By transfection of dynamin cDNA into mouse fibroblast L-cells, we showed it colocalized with some membranous organelles but not with microtubules. Our results show that dynamin is associated with membranous organelles in vivo, although a certain amount of dynamin also exists in the soluble fraction and is distributed diffusely throughout mature neurons. The data suggest that dynamin's fundamental role involves membrane trafficking in neurons in the central nervous system rather than in sliding microtubules as a motor protein.

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.

Centrosome repositioning immediately following karyokinesis and prior to cytokinesis.

The behaviour of the centrosome immediately following cell division in tissue culture cells has been investigated. We find that following karyokinesis, but preceding cytokinesis, sister centrosomes relocate from the spindle poles to a position adjacent to the intercellular bridge. This repositioning is accompanied by the appearance of a microtubule bundle that extends from the poleward region of the cell to the centrosome and increases in length as the centrosome approaches the intercellular bridge. Disruption of this bundle with colcemid interrupts centrosome repositioning. In contrast, centrosome repositioning persists in late mitotic cells grown in the presence of cytochalasin D. However, the position of the microtubule-centrosome complex within the cell is randomized suggesting that the path, but not the process, of centrosome repositioning is dependent on an intact actin filament network. This study points out, for the first time, that the complex migration of the centrosome preceding mitosis is paralleled by an equally complex set of events following cell division. We suggest that post-mitotic centrosome repositioning may play a role in ensuring that daughter cells have equal but opposite polarity and may reflect an interrelationship between the establishment of the interphase cytoskeleton and the completion of cytokinesis.

[The structure of the centromeric region and chromosome behavior in the anaphase after incubating murine cells with the fluorochrome Hoechst 33258]. / Struktura tsentromernogo raiona i povedenie khromosom v anafaze posle inkubatsii kletok myshi s fluorokhromom Khëkhstom 33258.

Mouse chromosome behaviour has been studied in the anaphase, after the cells (culture L) were incubated with fluorochrome Hoechst 33258, provoking the stretching of the chromosome centromeric regions. It was shown that in spite of marked structural changes in the centromere, the cells entered the anaphase and chromosomes preserved their ability to separate towards the spindle poles. At the same time, in the presence of Hoechst 33258, a reliable increase in the anaphase duration could be observed from 4.0 +/- 0.2 min in control to 6.0 +/- 0.5 min after a prolonged incubation of cells with the fluorochrome. Also, the increase in the frequency of chromosomal bridges in anaphase and the appearance of bridges in interphase cells were observed.

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Intracellular localization of the HLA class II gene products in transfected mouse L cells.

To investigate the impaired cell surface expression of human major histocompatibility antigen (HLA) in transfected L cells, we examined their intracellular localization by immunocytochemistry. HLA class II molecules produced in transfected L cells were mainly detected in the intracellular vesicles and in the nuclear envelope as granular precipitates. The results suggest that the intracellular transport of the newly synthesized molecules in transfected L cells is impaired at some point along the pathway from the rough endoplasmic reticulum (RER) to the medial-, trans-Golgi apparatus.

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.

A rapid method for screening vaccinia virus recombinants.

A rapid and small-scale method for screening vaccinia virus recombinants employing micrococcal nuclease is described. This protocol utilizes the differential sensitivity of cellular and viral DNA to the nuclease, which can be selectively activated by addition of Ca2+ and inactivated by elimination of Ca2+. Two to five micrograms of viral DNA can be obtained from one infected L cell plate (50 mm) after overnight incubation.

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.

Microcinematographic analysis of further fate of multinucleate cells arising after polyethylene glycol induced fusions of L cells.

Cell fusions were studied in continuous cell line L treated with polyethylene glycol (PEG). The progenies of 152 treated cells were studied by time-lapse microcinematography up to four generations. It was found that all the cells that remained mononucleate after treatment with PEG grew actively and their mitotic divisions were without abnormalities. On the contrary, only 60% of multinucleated (fused) cells divided mitotically during the filming period. We observed many disturbances in mitotic divisions of multinucleated cells. In most cases the multipolar divisions of these cells resulted in mononucleate daughters. In half of these cases the daughter cells fused to form again multinucleate cells. Nevertheless, both the total number of multinucleate cells and the average number of nuclei per cell were lowered after mitoses.

Detection of individual fluorescently labeled reovirions in living cells.

Reovirus serotype 1 (Lang) can be conjugated with rhodamine B or fluorescein isothiocyanate in a way that preserves viral infectivity. We have used epifluorescence microscopy to detect individual virions bound to the surface of cells and to follow in real time the early stages of reovirus infection in living cells. Following uptake of the virus into endocytic vesicles, the movement of these vesicles can be observed readily. The vesicle movement is inhibited by nocodazole or colchicine, consistent with previous findings that the movement of intracellular vesicles is often microtubule-based.