Involvement of cortactin and phosphotyrosine proteins in cell–cell contact formation in cultured bovine corneal endothelial cells

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell–cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including α-catenin, vinculin and cortactin, are localized at cell–cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell–cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y⁴⁶⁶, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY⁴²¹-cortactin was localized at the leading edge and pY⁴⁶⁶-cortactin at a perinuclear area. In confluent cultures both pY⁴⁶⁶- and pY⁴²¹-cortactin isoforms were localized at the cell–cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell–cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y⁴⁶⁶ and associated with the Triton-insoluble fraction.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.     

Ultrastructural evaluation of 8–16 cell human embryos cultured in vitro

The fine structure of eight human embryos cleaving in culture are described. Preovulatory human oocytes aspirated at diagnostic laparoscopies were fertilized and developed in vitro by methods which produced normal pregnancies. Three 8-cell and five 10–16 cell embryos showing delayed cleavage were fixed, serially sectioned and examined to determine whether the embryos were developing normally or otherwise.The organization of two 8-cell embryos was apparently normal while the other embryos showed varying aspects of abnormal development. Most blastomeres contained organelles normally present in human ova and their fine structure closely resembled those of comparable mammalian embryos. Nearly all the cellular components encountered in early mammalian embryos were observed. Certain morphogenetic changes were also noted during early cleavage involving nuclei, smooth endoplasmic reticulum and mitochondria.Both normal and abnormal features of the embryos are reported. Multi-nucleated blastomeres and partial fragmentation were commonly seen in abnormal embryos. The importance of ultrastructural evaluation of embryos in an in vitro programme is revealed in this investigation.     

Immunohistochemical localization of α1-acid-glycoprotein in human liver parenchymal cells

Summary ₁-acid glycoprotein, a major human serum glycoprotein was detected and localized in human liver parenchymal cells of a biopsy specimen. A heavy metal salt containing fixative was required to retain sufficient antigen determinants of ₁-acid glycoprotein in order to visualize this protein by the peroxidase-anti-peroxidase unlabeled antibody enzyme method.     

Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the “in vivo cryotechnique (IVCT)” followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60 s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.     

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.     

Production of PEX protein from QM7 cells cultured in polymer scaffolds in a Taylor–Couette bioreactor

In recent decades, many practical applications were developed with regard to the Taylor–Couette device, for example, reaction, filtration, extraction and bioreactor. In this study, the Taylor–Couette bioreactor was used to culture cells seeded in a biodegradable porous scaffold and produce PEX protein. Two different cell lines (NIH/3T3 and QM7) were seeded into PLGA sponges, which were fabricated using a solvent-free supercritical gas foaming method, and then cultured in the Taylor–Couette bioreactor. Cell proliferation was characterized using Quant-iT™ PicoGreen^® dsDNA assay and the results indicated that high mass transfer rate in the Taylor–Couette bioreactor enhanced cell proliferation. Qualitative distribution of live/dead cells was characterized using LIVE/DEAD^® Viability/Cytotoxicity assay and SEM and the results showed that cells cultured in static control mainly proliferated on the outer surface while the cells of Taylor-vortex bioreactor group could penetrate into the scaffold. The production yield of PEX protein, from QM7 cells transfected with pM9PEX, was quantified using PEX ELISA and the results showed a much higher PEX mass per scaffold for bioreactor than the control. As such, there is potential for the use of Taylor–Couette bioreactor in the mass production of PEX protein.     

Effect of tobacco extract on surfactant synthesis and its reversal by retinoic acid—role of cell–cell interactions in vitro

Tobacco induces oxidative stress in the alveolar epithelium and causes its damage. Retinoic acid (RA) has a cardinal role in alveolar cell growth, differentiation, and maturation. The aim of the study was to investigate the role of cell–cell interactions and whether RA could reverse the effect of tobacco extract on epithelial function as expressed by surfactant synthesis. For this, an in vitro model, which provides multiple cell type interactions, as seen in vivo, was used. We had used the major lung cell types, alveolar epithelial and mesenchymal cells represented by the cell lines A549 (human lung adenocarcinoma cell line), and human fetal lung fibroblast-1 (HFL-1) for developing the monoculture and co-culture systems and studied the effect of tobacco extract and retinoic acid. The effect of tobacco and retinoic acid both singly and in combination on proliferation and surfactant synthesis was analyzed. Retinoic acid induced proliferation and upregulated surfactant synthesis in monocultures and co-cultures. Tobacco extract at 100 μg/ml concentration decreased A549 proliferation and upregulated surfactant protein mRNA expression. In co-cultures treated with tobacco extract (100 μg/ml), retinoic acid (1 μM), regulated cell proliferation, and surfactant protein mRNA expression vis-à-vis the monoculture system. This clearly points to the fact that cell–cell interactions modulate the effect of additives or stimulants and help in assessing the in vivo combinatorial responses in vitro and that the retinoic acid effect is regenerative.     

Degradation of insulin in vitro by liver and epididymal adipose tissue from obese–hyperglycaemic mice

The insulin-degrading activity of liver supernatants and epididymal adipose-tissue homogenates from genetically obese–hyperglycaemic mice (ob ob) and their lean litter mates was studied by measurement of radioactive trichloroacetic acid-soluble degradation products of the insulin molecule. Optimum assay conditions for the decomposition of the hormone were devised. The properties of the degrading activity suggested the presence of enzymic insulin destruction in both the liver and epididymal adipose tissue. There was no difference in insulin degradation in liver samples from obese and lean mice when the results were related to the protein content of the supernatants. The epididymal adipose-tissue homogenates from obese mice displayed about eightfold higher degrading activity per unit of protein than did homogenates from lean animals. The physiological significance of this finding is discussed in the light of the increased fat depots, hyperphagia, raised serum insulin concentrations and increased insulin tolerance previously recorded in this strain of mice.     

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using “in vivo cryotechnique” (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid–Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.     

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.