Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes.

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.

A regulatory role of fibroblast growth factor in the expression of decorin, biglycan, betaglycan and syndecan in osteoblasts from patients with Crouzon's syndrome.

Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzon's syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzon's syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.

Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients.

During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.

Genetic expression profiling of six odontogenic tumors.

Odontogenic tumors are rare neoplasms arising from the odontogenic apparatus. We aimed to identify molecular characteristics associated with odontogenic tumorigenesis and malignancy. To this end, we investigated the expression level of human genes by using, for the first time in odontogenic tumors, the technique of expression profiling. Gene expression alterations common to all six odontogenic tumors were identified by the use of cDNA microarrays containing 19,000 human cDNAs. Statistical analysis on a subset of 4974 cDNAs present in the biopsies identified 506 distinct genes associated with the tumors (p-value < 0.01). Gene ontology analysis of the cellular processes which were differentially regulated in odontogenic tumors was accomplished by the use of a subset of 1409 annotated genes. Finally, 43 cDNAs differentiated the three malignant odontogenic tumors (ameloblastic carcinoma, clear cell odontogenic tumor, granular cell odontogenic tumor) from the three benign ameloblastoma biopsies (p < 0.01). The identified genes might help us better classify borderline odontogenic tumors.

TGFbeta isoforms and decorin gene expression are modified in fibroblasts obtained from non-syndromic cleft lip and palate subjects.

Interaction between extracellular matrix (ECM) and cytokines is thought to be crucial for palatal development. The localization of transforming growth factors (TGFalpha and TGFbeta isoforms) in craniofacial tissues suggests that they carry out multiple functions during development. In the present report, we studied TGFalpha, TGFbeta1, and TGFbeta3 expressions and their effects on ECM macromolecule production of normal and cleft palatal fibroblasts in vitro, to investigate the mechanisms by which the phenotypic modulation of fibroblasts occurs during the cleft palate process. The results indicated that, while TGFalpha mRNA was not evidenced in CLP or normal fibroblasts, a reduced TGFbeta1 hybridization signal was detected in CLP fibroblasts. In addition, these secreted more active TGFbeta3 than TGFbeta1, as evaluated in a biological assay. The CLP phenotype, which differed from the normal one because of its higher PG decorin expression and greater production of GAG and collagen, was further modified by the addition of growth factors. In fact, in CLP fibroblasts, TGFalpha and TGFbeta1 down-regulated PG decorin transcript, TGFbeta1 increased collagen and GAG in both cellular and extracellular compartments, and TGFbeta3 promoted secretory processes of cells. In conclusion, the data represent the first report in a human model in vitro that TGFbeta1 and beta3 are differently expressed and are correlated to the CLP phenotype. Thus, strength is given to the hypothesis that TGFbeta isoforms are the potential inducers of phenotypic expression in palatal fibroblasts during development and that an autocrine growth factor production mechanism may be responsible for the phenotypic modifications.

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1.

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

Coordinate effects of Concanavalin A on cytoskeletal organization, cell shape, glycosaminoglycan accumulation and exoglycosidase activity in chick embryonic cultured fibroblasts.

The effects of Concanavalin A on cell morphology, cytoskeleton arrangement and some metabolic activities of 11-day chick embryo fibroblasts were examined. In fibroblasts, Con A caused a dose-dependent round morphology and a change in tubulin, actin and alpha-actinin arrangement, whereas it did not modify 3H-thymidine incorporation. In addition, lectin stimulated more hyaluronic acid than sulphated glycosaminoglycan synthesis, affected glycosaminoglycan sulphation, and also reduced beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase and beta-glucuronidase activities.

[Standard parameters in gait analysis in an Italian multicenter case study]. / Parametri normativi dell'analisi del cammino in una casistica multicentrica italiana.

The study reports the results of a group who, in a multicentric trial, using a gait analysis laboratory Italian made, and a standard procedure, examined 127 normal subjects. The gait laboratory is composed of contacts to relieve the support on the ground, goniometric transducers with an articulated parallelogram, active sensors for cutaneous electromyography, patient unit for data collection and transmission, interface modules for signal reconstruction, software for data elaboration. All data was elaborated in order to give normative data for Italian population. There were no differences between right and left side, nor between male and female subjects. Values of the present study was compared with previous foreign literature and a critical comment is proposed.

Age-related changes in hyaluronate uptake by cultured fibroblasts.

Skin fibroblasts from chick embryos of 7 and 14 days incubation have been tested for their ability to bind, internalize and degrade [3H]-hyaluronan. Seven-day fibroblasts internalize and degrade a greater amount of labelled hyaluronan than 14-day cells. The implications of these findings in the regulation by the fibroblasts of extracellular matrix composition in glycosaminoglycans are discussed.

Involvement of the cytoskeleton in regulation of collagen synthesis and secretion.

11-day chick embryo skin fibroblasts administered in vitro with colchicine or cytochalasin B, have been investigated for collagen synthesis and secretion. The cytoskeleton, examined by confocal laser scanning immunofluorescence microscope, exibited in colchicine treated cells a clear loss of microtubules, in cytochalasin B treated cells a diffuse staining and patches of intracellular fluorescence caused by actin disassembly. Cytochalasin B treatment decreased endocellular type I collagen synthesis and the total uptake of 3H-proline, while enhanced collagen secretion. Colchicine did not interfere with the collagen secretion and the label uptake, whereas it increased endocellular collagen synthesis. In addition, both drugs induced an increase of the alpha2:alpha1 ratio of collagen chains. The results obtained suggest that the cytoskeleton controls not only the morphological organization of cell components, but also the organization of biochemical processes.

Exogenous glycosaminoglycans modulate extracellular and intracellular accumulation of newly synthesized glycosaminoglycans by embryonic chick skin fibroblasts.

The effects of exogenous glycosaminoglycans (GAG) on newly synthesized GAG accumulation in intra- and extra-cellular compartments of 17 incubation days chick embryonic skin fibroblasts have been examined. Exogenous GAG are able to act on both total GAG synthesis and secretion by embryonic fibroblasts and on the relative concentration of individual GAG. A decline in hyaluronic acid and an increase in chondroitin sulfate plus dermatan sulfate in the cellular compartment for all GAG administered have been detected. There was also similar behaviour in the extracellular compartment after sulfated GAG administration.

[Analysis, by lectin fluorescence, of the cell surface of embryonal fibroblasts cultivated in vitro]. / Analisi, per mezzo di lectine fluorescenti, della superficie cellulare di fibroblasti embrionali coltivati in vitro.

Cultured fibroblast derived from 7 and 14 days chick embryonic skin were tested with three fluorescent lectins (Con A, WGA and SBA). The result obtained shown that the percentage of cells that bind WGA decrease from 7 to 14 days, supporting the presence of an age-dependent heterogeneity at cell surface level. In both cellular population only the 50% of the cell bind to SBA, suggesting a more subtle heterogeneity into the same population.

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.

Cyclosporin A, effect on cytoskeleton and glycosaminoglycans in human gingival fibroblasts. Immunohistochemical and biochemical evaluation.

The effect of Cyclosporin A (CyA) in culture of human gingival fibroblasts was assessed. Extracellular and intracellular glycosaminoglycans decrease in treated cells. The ratio of non sulphated over sulphated GAGs (glycosaminoglycans) increased only in the medium. Cytoskeleton organization of treated fibroblasts was also studied by immunohistochemical means and by ultrastructural observations. Minor alterations were documented in the structure and distribution of cytoplasmic organelles. The organization of tubulin and vimentin filaments did not change in Cy A treated cells either. This study confirms the capability of Cyclosporin A to induce alterations of GAG production and synthesis with no interference in cytoplasm organization. Normal pathway GAG secretion is suggested.

Heterogeneity of fibroblasts derived from human free and attached gingiva. Glycosaminoglycan synthesis and effects of phenytoin (PHT) treatment.

Two fibroblast populations derived from free and attached gingiva (FGF, AGF) have been compared in cell culture. They exhibited the same morphology and similar cytoskeletal staining patterns, but were different in glycosaminoglycan (GAG) synthesis. FGF released larger quantities of GAGs than AGF in the medium. The extracellular accumulation of hyaluronic acid was higher in FGF than in AGF, whilst the reverse pattern was observed intracellularly. In the case of sulphated GAGs the extracellular concentration, compared to HA, was higher in AGF, while the intracellular concentration was higher in FGF. The two cell populations responded differently to phenytoin (PHT) administration. PHT treatment increased the proportion of intracellular sulphated GAGs in AGF and of extracellular sulphated GAGs in FGF.

Comparative effects of TGFbeta on proliferation of 7- and 14-day-old chick embryo fibroblasts and lack of involvement of the ODC/PA system in the TGFbeta signaling pathway.

The growth regulatory activity of transforming growth factor beta (TGFbeta) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of 3H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFbeta treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFbeta proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFbeta on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFbeta signaling.

Concanavalin A affects glycosaminoglycans cellular and extracellular accumulation in cultured embryonic fibroblasts.

Incorporation of 3H glucosamine and 35SO4 into glycosaminoglycans and proteoglycans produced and secreted by 7, 11 and 14 day chick embryo fibroblasts in vitro after concanavalin A treatment has been determined. Lectin differently affects 3H and 35SO4 incorporation. It enhances 3H labelled GAG accumulation in both cellular and extracellular compartments. Total incorporation of 35SO4 remains unchanged whereas the intracellular one is stimulated and the extracellular is reduced. All the effects are more relevant in the early stages of development. HA and PG cellular and extracellular accumulation seems to be independently regulated.

Involvement of polyamines in the action of transforming growth factor beta and interleukin-1 on cultured chick embryo fibroblasts.

In this study we have examined the relationship between growth factor-induced proliferation and ODC/polyamine levels. TGF beta promotes cell growth and enhances [3H]-thymidine incorporation in chick embryo fibroblasts maintained in a serum-depleted medium. The action on DNA synthesis declines in the second day of treatment. IL-1 does not affect proliferation or [3H]-thymidine incorporation either when it is added alone or in combination with TGF beta. The response of the cells to TGF beta is associated with a significant stimulation of ODC activity and Put, Spd levels together with an enhancement of the Spd/polyamines ratio. IL-1, which does not act on cell proliferation, fails to activate ODC and to increase polyamine levels, thus indicating that the ODC/polyamine system is most likely to be an important link in the chain of events that leads to growth factor-induced proliferation.

Age related and lectin influenced changes of exoglycosidases activity in cultured chick embryonic skin fibroblasts.

beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-galactosaminidase and a beta-D-galactosidase activity was determined in untreated and lectins (ConA, PNA, SBA and WGA) treated chick embryonic skin fibroblasts at two incubation stages. Activity of all three glycosidases increased between 7 and 14 incubation days. ConA and WGA affected the levels of enzymatic activity; while SBA and PNA were uneffective. We discuss these findings in relation to a possible role of glycosidases in controlling mesenchymal GAG turnover.

Scanning electron microscopic observations on the epithelium of the human prostatic urethra.

The human prostatic urethra has been investigated by means of scanning electron microscopy. On the posterior wall of the urethra, the seminal colliculus with the orifices of the ejaculatory ducts is clearly detectable. The upper portion of the prostatic urethra shows a typical transitional epithelium with large superficial cells of a ruffled appearance. In the lower portion of the organ (underneath the openings of the ejaculatory ducts), the apical pattern of the cells varies considerably. Four main aspects are recognizable: apices provided with microvilli, dome-shaped apices with an almost smooth surface, large apices with labyrinthic microplicae and ciliated apices. Also, apices showing intermediate characteristics can be noted. The functional significance of the morphological patterns as well as the possibility of a transition among the various types of surface structures are discussed.