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ABSTRACT: The purpose of the present study was to establish a method for light microscopical immunohistochemical localization of the small G protein RhoA on specimens treated and embedded for routine transmission electron microscopy. There are advantages in antigen immunolocalization on resin semi-thin sections compared to cryostat or paraffin sections: the preservation of morphological details, the well-defined immunoprecipitate localization and the possibility to correlate the immunohistochemical results with those obtained by electron microscope on neighbouring sections. These advantages are particularly useful for the subcellular localization of low molecular weight proteins such as RhoA, a small G protein able to cycle from the inactive cytoplasmic form to the plasma membrane-bound active form.
Micron 02/2004; 35(4):287-96. · 1.53 Impact Factor
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ABSTRACT: The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor alpha (TGFalpha), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates. The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys. TGFalpha messenger ribonucleic acid, evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines. Large amounts of TGFalpha peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells. RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines). The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates. This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFalpha found in significant amounts only in the SK-NEP-1 cell line.
In Vitro Cellular & Developmental Biology - Animal 05/2001; 37(4):251-8. · 1.31 Impact Factor
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ABSTRACT: The small G protein Rho subfamily controls several cellular events such as growth, movement, proliferation and differentiation by rearranging actin and cytoskeleton proteins. Most of these effects are mediated by the activation of growth factor and extracellular matrix molecule receptors, suggesting a role for Rho molecules in the transduction pathway of these receptors. Despite the importance of Rho peptides in fundamental cellular events, data on their subcellular immunolocalisation are sparse: here we investigated the expression and subcellular localisation of RhoA in resting (cultured on plastic) and activated (Matri-cell or hepatocyte growth factor) MDCK cells by immunoperoxidase and immunogold techniques. Resting MDCK cells contain detectable amounts of RhoA mainly localised in the cytoplasm; RhoA expression is significantly enhanced by Matri-cell substrates that promote translocation of RhoA at the membrane level. This enhancing effect is reduced after exposure to hepatocyte growth factor.
Cellular and Molecular Life Sciences CMLS 01/2001; 57(13-14):1990-6. · 6.57 Impact Factor
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ABSTRACT: Laminin, an extracellular matrix molecule (EMM) widely expressed in the basal laminae, interacts with specific membrane receptors among which the integrin molecules are the best known. During embryo development laminin is the first synthesized EMM and plays a significant role in the morphogenesis of organs in which epithelial-mesenchymal interactions and branching take place. The present study describes the distribution of laminin and of beta1 integrin receptors during the very early stages of human kidney development. The observations were carried out on paraffin sections of human embryos ranging between the 4th and the 7th gestational week. Laminin was detected within the basement membranes of mesonephric duct, vesicles, glomerular vessels and celomic epithelium. The metanephric anlage reacted with anti-laminin immunoglobulins in the basement membrane underlying the ampullae and in few blastemic cap cells. Low levels of beta1 integrin reactivity were found in both the mesonephric and metanephric structures. This study provides for the first time data about the distribution of laminin and beta1 integrin in the early stages of human renal organogenesis suggesting a key role for these molecules in the epithelial-mesenchymal interactions necessary for kidney development.
Nephron 02/1999; 81(3):289-95. · 13.26 Impact Factor
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U Benelli,
G Bocci,
R Danesi,
A Lepri,
N Bernardini, F Bianchi,
M Lupetti,
A Dolfi,
A Campagni,
C Agen,
M Nardi,
M Del Tacca
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ABSTRACT: The purpose of this study was to evaluate the inhibitory activity of the heparan sulfate suleparoide on vascular cell growth in vitro and angiogenesis in vivo. Human HUV-EC-C endothelial cell proliferation and microvessel sprouting from cultured rat aortic rings were assayed by the bioreduction of 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. The inhibition of the neoforming capillary network in the chorioallantoic membrane of chick embryo (CAM) was evaluated by agarose disks containing suleparoide and applied on the CAM surface. AgNO3/KNO3 injury was used to induce corneal neovascularization and to evaluate the therapeutic effect of topical suleparoide, while the involvement of bFGF in angiogenesis was evidenced by immunohistochemistry of corneal tissue. Quantitation of angiogenesis in the CAM and the cornea was accomplished by image analysis. Suleparoide dose-dependently inhibited HUV-EC-C cell proliferation (50% inhibitory concentration [IC50], 197.5+/-15.2 microg ml-1) and reduced microvessel sprouting in vitro (IC50, 351+/-22 microg ml-1). Likewise, suleparoide 150 microg in agarose disks produced an avascular area of 19.7+/-2.7% of the total area of the CAM (P<0.05 as compared to controls). bFGF levels were significantly enhanced in the cornea after AgNO3/KNO3 injury, and the increase appeared to be time-dependent (25.6+/-1.8 and 43.2+/-7.4%, vs. uninjured controls after 24 hr and 48 hr, respectively, P<0.05). Suleparoide 4.8 mg eye-1 day-1 for six days reduced the length of blood vessels and the area of the cornea infiltrated by them (59.6+/-7.4% decrease vs. controls, P<0.05). These results demonstrate that suleparoide is an active agent against angiogenesis and suggest that the therapeutic effect of the drug could be of value to treat corneal neovascularization.
Experimental Eye Research 08/1998; 67(2):133-42. · 3.26 Impact Factor
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ABSTRACT: The distribution of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and EGF/TGF alpha receptor were studied by means of immunohistochemical methods starting from the very early stages of human embryonic kidney development. Mesonephros and metanephros were examined in order to detect immunoreactive staining in serial sectioned embryos and fetal kidneys. Anti-EGF immunoprecipitates were found in the S-shaped mesonephric vesicles of 6-week old embryos as well as in the mesonephric duct albeit with a lower degree of reactivity. Intense reactivity was observed in the metanephros within the blastemic caps of the same gestational period; the reaction was weaker within the ureteric bud branches. Bowman's capsule, proximal tubules, and collecting ducts were also reactive in the fetal kidney to varying degrees. The distribution of TGF alpha reactivity in the mesonephros was similar to that observed for EGF but with a lower intensity. In contrast, there was no reactivity in the metanephros, at least during the embyronic periods examined. By the 11th week of gestation, an intense reactivity for TGF alpha polipeptide was shown in the fetal kidney at the level of the proximal tubules and Bowman's capsule; distal tubules as well as all urinary structures from the collecting ducts to the pelvis were less reactive. Finally, EGF/TGF alpha receptor reactivity was identified by the 6th week of development, being more intense in the mesonephros at the level of the mesonephric duct cells. In the metanephros, the ureteric bud-derived branches were reactive, whereas most of the blastemic tissue did not stain. By the 11th week, only the collecting ducts and the remaining urinary structures contained reaction products: Reactivity was distributed to the tissues originating from the ureteric bud branching. Taking into account recent advances in knowledge about the biology of growth factors, the hypothesis is proposed that the secretory components (vesicles, glomerulus, and tubules) of renal anlagen might release the growth factors while the cells of the urinary tract (i.e., collecting duct, pelvis, etc.) may be their targets.
Developmental Dynamics 08/1996; 206(3):231-8. · 2.54 Impact Factor
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ABSTRACT: Type I-iodothyronine monodeiodinase (type I-MD) is abundant in thyroid tissue and contributes to the generation of T3 secreted by the gland. The availability of a specific antibody against rat type I-MD (type I-MD Ab) allowed us to directly identify this enzyme in rat thyroid glands, and in a differentiated strain of rat thyroid cells maintained in continuous culture (FRTL-5 cells). FRTL-5 cells maintain many differentiated functions of thyroid cells, including the expression of TSH receptor and thyroid peroxidase. Using an immunohistochemical technique on rat thyroid sections, a clear staining for type I-MD was demonstrated in follicular cells. The degree of immunoreactivity was greater in small follicles containing little amounts of colloid compared to large follicles lined by functionally inactive cells. Using immunofluorescence (IFL), a strong staining for type I-MD was observed in FRTL-5 cells grown in medium containing TSH. Both in vivo and in culture the staining for type I-MD was localised in the cytoplasm of thyroid cells, while nuclei were negative. Interestingly, no surface staining was shown when viable FRTL-5 cells were submitted to the same IFL procedure. TSH deprivation for 7 days was followed by the disappearance of type I-MD. Immunoreactivity for type I-MD was recovered by addition of TSH, forskolin or thyroid stimulating antibody (TSAb) to TSH-deprived FRTL-5 cells. The effect of TSH was prevented by cycloheximide. There was no induction of type I-MD when IGF-I was added to FRTL-5 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular and Cellular Endocrinology 05/1995; 110(1-2):195-203. · 4.19 Impact Factor
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ABSTRACT: The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described. The present report evaluates the expression of the mdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching. Results show that the thymic cells are positive from day 12 to the end of the observation period. In contrast, mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life. Possible relationships between the expression of mdr and the development of T and B lymphocytes are discussed.
Experientia 03/1995; 51(2):137-40.
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Transplantation Proceedings 07/1994; 26(3):1127. · 1.00 Impact Factor
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ABSTRACT: Vascularization of the cornea occurs in many pathological conditions and can result in loss of visual acuity. It is also thought that vascularization predisposes the cornea to reject grafts by facilitating the detection of foreign antigens in donor material. A rat corneal assay for angiogenesis was adopted in the present study to evaluate the possible angiostatic activity of a low molecular weight heparan sulphate (LMW-HS). Corneal lesions were induced by chemical cauterization at 2 mm from the corneoscleral limbus. Rats were randomized to receive two drops/eye four times daily, for 6 days, of a solution of LMW-HS in vehicle (2.5% carboxymethylcellulose), heparin, heparin plus hydrocortisone, or vehicle alone. After a 6 day-treatment period, the eyes were perfused with india ink and the degree of neovascularization was evaluated. In rats treated with vehicle alone a dense vascular network extending from the corneoscleral limbus to the cauterized site was observed; on the contrary, a markedly reduced vascular network was evidenced in animals treated with LMW-HS. The distribution of basic fibroblast growth factor (bFGF) in the cauterized cornea was also evaluated by using an immunohistochemical method. A marked bFGF immunoreactivity was demonstrated in corneal epithelium and stroma of control rats 12-48 hours after the cautery. These results lead to the assumption that LMW-HS could be used in ophthalmology to inhibit corneal neovascularization.
Journal of ocular pharmacology 02/1994; 10(1):273-80.
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ABSTRACT: The technique whereby islets were isolated from the pancreas of chicken and their in vitro histological and functional characterization are described in this paper. Our isolation procedure consisted of two steps: initially the pancreas removed from the chicken was perfused with a 2% solution of collagenase and enzymatic digestion was then carried out using the same solution. After this, density gradient separation was performed on the digested tissue, by means of differing Histopaque solutions: at the end of the separation, the islets were studied by light microscopy after treatment with diphenylthiocarbazone, which selectively stains beta-cells, and by scanning and transmission electron microscopy. The results reported here show the pattern closely resembled that encountered when islets were studied in situ. The beta-cells of the islets proved in vitro to preserve their functional capability of producing insulin even after stimulation with glucose or arginine.
Tissue and Cell 01/1994; 25(6):817-24. · 1.04 Impact Factor
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ABSTRACT: Multidrug resistance (MDR) is frequently found in haematologic malignancies. It has been shown that MDR is often related to the expression of a membrane glycoprotein (P-170) which actively pumps many hydrophobic agents out of the cells. Previous electron microscopic investigations revealed morphological differences between P-388 resistant and P-388 sensible cell membranes, but the modulation of the membrane morphology seems to be related to the tumor-cell environment. In order to establish if morphological differences exist between sensitive and resistant cells, both sensitive and resistant strains from three different cell lines were studied by scanning electron microscopy: human leukaemia CEM and vinblastine resistant cells (CEM/VBL100), human breast cancer MCF-7 and mice leukaemia P-388 with the doxorubicin resistant strains (MCF-7/DX and P-388/DX, respectively). The surface of the membranes of the sensitive cells was regular, unlike the resistant ones which proved to be irregular, endowed with long villus-like processes or numerous folds and ruffles. The addition of albumin to the culture medium induced a shift from the resistant to the sensitive phenotype, thus suggesting that the P-388/DX morphology may be linked to the concentration of protein in the culture medium. Exposure to DX, verapamil (VRP) or monoclonal antibody against P-170 (mAb-57) did not modify the surface of the resistant strains, demonstrating that surface irregularities are probably not linked to P-glycoprotein function. Blasts from four P-170 positive leukaemic patients were also analyzed: an irregular shape was always found.
Cellular and molecular biology 08/1993; 39(5):543-51. · 0.98 Impact Factor
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ABSTRACT: The esophagus of the turtle, like the mucosal surfaces in other species, contains variously sized areas of lymphoid infiltration. The tunica propria and the surface epithelial layer of this area are invaded by the lymphoid cells. The features of the layer of epithelial cells which cover the lymphoid infiltrations are of a special kind: they do not possess vibratile cilia and are able to take up materials flowing into the lumen. The present paper contains further information concerning lymphoid infiltration obtained by histological and histochemical methods. The epithelial layer covering the lymphoid infiltrations is composed of cells with irregularly distributed microvilli, ciliated cells and mucous-secreting cells. After administration of silica and colloidal carbon, the microvillar epithelial cells proved to have these substances inside them, thereby accounting for the pinocytotic activity. The absorbing epithelial cells were not damaged by silica which is a macrophage-toxic agent, while the underlying macrophages are damaged. These results are compared with the features of lymphoid infiltration associated cells in various organs and animals; the hypothesis is proposed that these cells in the esophagus of turtles may originate from the covering epithelial cells.
Tissue and Cell 07/1993; 25(3):389-97. · 1.04 Impact Factor
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ABSTRACT: The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in 3H-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 microM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 micrograms ml-1 produced a modest reduction in 3H-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 micrograms ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 micrograms mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 micrograms ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth.
British Journal of Cancer 07/1993; 67(6):1209-16. · 5.04 Impact Factor
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ABSTRACT: Doxorubicin (DXR), an anthracycline antineoplastic drug, is mainly metabolized to the C-13 dihydroderivative doxorubicinol (DXR-ol), which displays cytotoxic activity on various cell lines. To better characterize the cytotoxic activity of this metabolite, we have studied the effect of DXR (0.1-10 micrograms/ml) or DXR-ol (1-100 micrograms/ml) on the transformed fibroblast cell line V79/AP4 by means of the clonogenic assay, cytofluorescence, and light and electron microscopy. Both DXR and DXR-ol displayed a dose-dependent inhibition of colony formation with an IC50 factor DXR-ol/DXR of 19.5. A striking nuclear fluorescence was observed after DXR but not after DXR-ol. A low number of mitoses and a decrease in nucleoli staining affinity were the most evident alterations induced by DXR. Electron microscopy showed both nuclear and cytoplasmic changes in DXR treated cells: nucleolar segregation, cytoplasmic vacuoles, and mitochondrial swelling with dense needle-shaped material were observed. Exposure to formic acid confirmed the calcific nature of the mitochondrial bodies. Only the highest dose of DXR-ol brought about nuclear and cytoplasmic ultrastructural changes similar to those induced by DXR. Our data describe new in vitro findings on the cytotoxicity and morphological alterations induced by both DXR and DXR-ol, with a lower activity of DXR-ol against V79/AP4 fibroblasts.
Experimental and Molecular Pathology 01/1992; 55(3):238-50. · 2.42 Impact Factor
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ABSTRACT: The vitamin D3 metabolite 1,25(OH)2D3 is probably involved in B lymphocyte ontogeny. We therefore determined the distribution of the 1,25(OH)2D3 receptor in the bursa of Fabricius and spleen cells of 7-day-old chicks, by immunohistochemistry using a monoclonal antibody against the chick intestinal cell 1,25(OH)2D3 receptor. The bursa cells of young (7-day-old) chicks contained large amounts of receptor while the spleen cells did not. The bursa cells of older (35-day-old) chicks contained fewer receptors, but the number of receptors in the spleen increased.
Experientia 09/1991; 47(8):838-41.
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ABSTRACT: The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmosomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.
Experientia 11/1990; 46(10):1060-3.
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ABSTRACT: The morphological relationships present in the areas of lymphoid infiltration found in the esophagus in Chrysemys scripta elegans were studied by the use of light and transmission electron microscopy. These infiltrations are widespread and can be observed in the lamina propria and the covering epithelium. Electron microscopy reveals the presence of epithelial cells with differing characteristics at the apex of the lymphoid infiltrations. The introduction of horseradish peroxidase (HRP) into the esophageal lumen revealed that these cells possess a micropinocytotic activity. On the basis of the morphological and experimental results obtained, a hypothesis is advanced of the existence, also in the turtle, of a local system of uptake of extraneous material, which induces local and systemic immunological processes.
The Anatomical Record 06/1990; 227(1):104-10.
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ABSTRACT: The development of the bursal follicle and the appearance of the follicle-associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of follicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surrounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and a number of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that have closed under the mesenchymal nodule become stratified and form the REp cells; they become star-shaped because the medullary-lymphoid cells grow between them. Finally, the cortex is formed, possibly as a result of the migration of medullary cells before they peripheralize. It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.
American Journal of Anatomy 04/1990; 187(3):287-302.
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ABSTRACT: Adenosine deaminase, a purine metabolic enzyme, was studied in lymphoid tissues of the developing chicken in order to evaluate whether enzyme activity is related to development of the immune system in birds in the same way as for mammals, in which adenosine deaminase is essential for lymphocyte differentiation, especially for the T-cell lineage. Enzyme activity was assayed in thymocytes and bursal lymphocytes at different times during chicken development ranging from the 17th day of embryonic life up to the 50th day after hatching. Adenosine deaminase activity was significantly higher in the bursa than in the thymus, regardless of whether such an activity was expressed per mg protein or per 10(8) cells; moreover, no substantial difference in the relative levels of adenosine deaminase was observed in thymocytes at the various stages of thymus development studied. Significant changes in enzyme activity, however, were found in bursal lymphocytes in which different amounts of adenosine deaminase appeared to be related to definite stages of bursal development and to specific immunological responsiveness of B lymphocytes to intravenously injected antigens. Therefore, if adenosine deaminase does play a role in the functional maturation of the immune system in birds, such a role appears to be related to the differentiation of the B- rather than the T-cell lineage.
Developmental & Comparative Immunology 02/1990; 14(1):95-104. · 3.27 Impact Factor
