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To investigate the involvement of extracellular matrix (ECM) in folliculogenesis in the sheep, parallel changes in ECM components and key steroidogenic enzymes were studied by quantitative immunohistochemistry and immunoblotting during follicular growth and atresia. Growth of ovarian follicles from 1 to 5 mm in diameter was characterized by a progressive increase in P450 cholesterol sidechain cleavage levels in both thecal (p < 0.001) and granulosa cells (p < 0.001), an increase in P450 aromatase levels in granulosa cells of follicles larger than 3.5 mm (p < 0.001), and an increase in levels of P450 17 alpha-hydroxylase C17,20 lyase (P450(17 alpha)) in the theca interna. In addition, during follicular growth, a change in localization of cells expressing P450(17 alpha) within the theca interna was observed, positive cells being sparse within the theca interna of small follicles and specifically located close to the basal laminae in large follicles. In parallel, follicular growth was associated with an increase in levels of type I collagen in granulosa cell layers (p < 0.01) and an increase in levels of fibronectin (p < 0.05), particularly the specific ED-A alternatively spliced variant of fibronectin, in the theca externa. Follicular atresia was characterized by a loss of P450 aromatase in granulosa cells (p < 0.001) and a decrease in levels of P450(17 alpha) in the theca interna (p < 0.05). Simultaneously, levels of fibronectin (p < 0.05), particularly the ED-A variant of fibronectin, decreased in the theca externa of atretic follicles. Within the wall of granulosa cells, levels of fibronectin (p < 0.05), laminin, type IV collagen, and heparan sulfate proteoglycans strongly increased during follicular atresia. Overall, these results show that follicular growth and atresia were associated with distinct changes in levels of ECM components, suggesting that ECM components may play a role in the regulation of proliferation, differentiation, and apoptosis of follicular cells.

General characteristics of female reproductive activity, such as seasonality, cyclicity and triggering of ovulation differ widely among mammals, but common mechanisms underlie ovarian function. In all mammals, follicles begin to grow from a pool of primordial follicles constituted early in life, continuously throughout the life of the female. Follicular development involves two phases. In a first phase (basal follicular growth), follicles grow slowly and follicular growth rate is tightly related to proliferation of granulosa cells. Basal follicular growth is mainly under the control of growth factors of paracrine origin. In these follicles, FSH may exert an indirect mitogenic effect on granulosa cells by enhancing expression of growth factors or growth factor receptors. In a second phase (terminal follicular growth), follicular growth is rapid and occurs by enlargement of the antrum. In addition, it is accompanied by important changes in differentiation of follicular cells. Terminal follicular development is strictly dependent on gonadotrophins. FSH plays determinant roles in enhancing granulosa cell differentiation and survival. These actions are mediated or modulated in an important way by paracrine factors, particularly steroids and growth factors. LH stimulates steroidogenesis in theca cells and sustains terminal maturation of granulosa cells in preovulatory follicles. Follicular growth, atresia and ovulation are accompanied by important tissue remodelling processes, which are under the fine control of proteinases and inhibitors of proteinases. In particular matrix metalloproteinases and their inhibitors are probably involved in the control of rapid terminal follicular growth and regression of atretic follicles as well as in follicular rupture at ovulation.

Introduction: Bazex paraneoplastic acrokeratosis remains a therapeutic challenge when the cancer cannot be treated. We report the third patient in which a complete clearance of the lesions was obtained with oral acitretine., Case Report: A 67 year-old patient had Bazex paraneoplastic acrokeratosis. Despite a complete cancer screening, no cancer was found. A cervical lymph node metastasis was treated with surgery and radiotherapy. Because the cutaneous lesions got worse after this treatment, the patient received oral acitretine. A complete clearing of the lesions was observed within 2 months., Discussion: Only 2 similar cases have been reported to our knowledge. The comparison with these 2 cases supports the efficacy of oral retinoids in this disease. The search and the treatment of the cancer remain compulsory., Conclusion: In some definite cases, oral retinoids can be proposed as a treatment of cutaneous lesions in Bazex paraneoplastic acrokeratosis.

Neutral endopeptidase (Endopeptidase 24.11; NEP; neprilysin), an integral membrane protein, and villin, a major microvillar cytoskeletal actin-binding protein, are both typically associated with brush border epithelia. In this study, cRNA probes were hybridized in situ to investigate the expression of NEP and villin genes in embryo and adult mouse enterocytes. During development, villin mRNAs were easily detected in the immature digestive tract well before establishment of the brush border. In 17-day-old embryos, a transient elevation of villin mRNA occurred just prior to a dramatic increase in microvilli length and density. NEP only appeared by day 17 as the embryonic gut began to become functional. It therefore appears that the onset of transcription of specialized cytoskeletal proteins from the brush border preceded that of intrinsic membrane-bound enzyme from microvilli. In the adult intestinal fold, both mRNAs were expressed along the whole length of the villus with maximal expression at its base. In contrast, both proteins were uniformly expressed along the whole crypt-villus axis. Quantitative analysis revealed an asymmetric intracellular distribution of both mRNAs that were differentially polarized in the apical cytoplasm of enterocytes.

Immature inflorescences of coconut belonging to three different genotypes were cultured on a solid medium supplemented with activated charcoal (2%) and a range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (from 1.5 to 3.5 × 10(-4)M). Globular white callus formed from immature floral meristems, depending on inflorescence age and 2,4-D concentration. Acquisition of embryogenic competence is described histologically. Somatic embryos presented a functional bipolar organization with a completely differentiated shoot meristem which is reported here for the first time in coconut tissue culture. Embryo maturation allowed reliable plant regeneration of this in vitro recalcitrant species. Details are given of exogenous hormonal requirements for the acquisition of embryogenic competence and embryo maturation.

Caco-2 cells were cultured on permeable supports. At confluence the minute-by-minute net water movement (Jw) was automatically recorded. Simultaneously, unidirectional [14C]mannitol, 22Na+, and/or 36Cl- fluxes and transepithelial resistances were measured. The water and mannitol permeabilities went progressively down between 9 and 16 days after seeding and then stabilized. In this last condition the hydrostatic permeability coefficient (Phydr) was 2.67 +/- 0.31 cm s-1 while the osmotic permeability coefficient (Posm) was 0.0017 +/- 0.0004 cm s-1. Phydr but not Posm was dependent on the temperature and on the presence of Na+ in the medium. A net secretory Jw was observed 16 days after seeding, in the absence of any osmotic, hydrostatic or chemical gradient. This secretory Jw was associated with net Cl- (1.43 +/- 0.43 muequiv h-1 cm-2) and Na+ (1.05 +/- 0.35 muequiv h-1 cm-2) secretions. Amiloride reduced, in open-circuit conditions, both Na+ and Cl- apical to basal fluxes, thus enhancing the net Na+ and Cl- exit. Acidification of the medium (pH 6.2) reversibly increased water and mannitol permeabilities in 10-day-old cultures. In 16-day-old cultures the same shift in medium pH did not change mannitol permeability, while stimulating water secretion. These results, obtained in the absence of supracellular structures (villae, crypts) and subepithelial components (muscular, vascular and conjunctive tissues) indicate that paracellular and transport-associated water pathways are sensitive to changes in the pH of the medium in Caco-2 cell layers.

The actin-binding protein villin induces microvillus growth and reorganization of the cytoskeleton in cells that do not normally produce this protein. Transfection of mutagenized villin cDNAs into CV-1 cells was used to show that a conserved, COOH-terminally located cluster of charged amino acid residues (KKEK) is crucial for the morphogenic activity of villin in vivo. In vitro experiments with a 22 amino acid synthetic peptide corresponding to this region of villin provide evidence that this motif is part of an F-actin-binding site that induces G-actin to polymerize. Chemical cross-linking of actin to this peptide, the effects of amino acid substitutions in peptides, and the behavior of villin variants further corroborate the participation of the KKEK sequence in actin contacts.

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.

The female presumptive genital epithelium ofTenebrio molitor shows two stages of determination. 1) An essential determination which appears in the larva, but which must occur during the embryonic period. 2) A mosaic determination which is reached within twenty four hours before metamorphosis. The presumptive genital epithelium is able to complete its organogenesis normally after becoming competent and being submitted to the hormonal conditions of metamorphosis. The acquisition of competence appears to be linked to a process of growth.

A glycolipid extract was prepared from an SV40-transformed hamster cell line (EH-SV) according to the Folch partition procedure. The glycolipids from the aqueous layer were incorporated in liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1:1:2 by weight). This liposomal preparation was inoculated in Syrian hamsters to raise immune sera. The sera were absorbed with trypsinized "normal" hamster cells (EH-N) and tested on various cell lines by the indirect immunofluorescence technique. When used for staining living cells, the immune serum produced a distinct cell-surface fluorescence with SV40-transformed cell lines regardless of the cell origin (e.g., rat or hamster). No reaction was observed with heterologous Py-transformed cell lines, spontaneously transformed cells, or sera from non-immunized hamsters. When used for staining acetone-fixed cells, the antiglycolipid serum reacted specifically with a thermostable antigen in the nuclear envelope and the cytoplasm of SV40-transformed cells. The sera lack interfering SV40 T reactivity. The results indicate the presence of related SV40-specific glycolipid antigen(s) in the plasma membrane, the nuclear membrane and probably other endomembranes of SV40-transformed cells.

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed hamster cell lines. Immunization with this virus protected hamsters against challenge with SV40-transformed cells (TSV5-cl2). This protection was obtained regardless of the source of the SV40-transformed cells (e.g. cat, rat, hamster) used to produce VSV, and was therefore associated with the SV40 tumor-specific transplantation antigen (SV40-TSTA). Furthermore, when grown on spontaneously transformed cell lines or on cells transformed by a different oncogenic DNA virus, such as polyoma virus, the VSV failed to protect against the SV40-induced tumor. It was concluded that the SV40-TSTA activity of purified VSV is due to the incorporation of SV40-TSTA within the viral envelope. When VSV was treated with proteolytic enzymes (bromelain, trypsin) no loss of TSTA-induced tumor rejection was observed, although VSV had lost its ability to induce virus-neutralizing antibody. This clearly demonstrates that the TSTA activity is not related to the viral spikes. Phospholipase C suppressed the TSTA activity but neutralizing activity was still detectable in the anti-VSV sera. The results presented here demonstrate that the protection afforded by VSV is highly specific. It is particularly interesting that SV40-TSTA activity may be conveyed by the lipid core of the viral envelope.

The function of villin, an actin-binding protein, has been investigated by transfecting fibroblasts with cloned human cDNAs encoding wild-type villin or functional villin domains. Synthesis of large amounts of villin induced the growth of numerous long microvilli on cell surfaces together with the redistribution of F-actin. These microvilli contained a cytoskeleton of F-actin, and their appearance was frequently accompanied by the disappearance of stress fibers. The complete villin gene sequence was required to exert its morphogenic effect. Villin lacking one actin-binding domain (113 amino acids), located at its carboxyterminal end, did not induce growth if microvilli or stress fiber disruption. Our results indicate that villin plays a key role in vivo in the morphogenesis of microvilli.

HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this report to be a model system for the study of human goblet cell differentiation and mucin secretion. Grown in the absence of glucose, these cells formed homogeneous epithelial monolayers of columnar cells with typical goblet cell morphology. Differentiation occurred on uncoated glass; laminin, fibronectin, or collagen type I or IV did not enhance differentiation. HT29-18N2 cells grown on uncoated or matrix-coated permeable filters formed differentiated monolayers, but mucin granules within some of these cells polarized along intraepithelial lumens. Polyclonal antibodies raised against purified human colonic mucin, and also a monoclonal antibody against a protease-sensitive epitope of human colonic mucin, stained secretory granules of all differentiated goblet cells within N2 cell monolayers but did not stain predifferentiated goblet cells lacking large secretory granules. Monoclonal antibodies against specific carbohydrate sequences of human mucins also failed to stain N2 cells before differentiation, but recognized varying fractions of differentiated N2 goblet cells. Autoradiographic visualization of radiolabeled glycoproteins demonstrated transport and secretion of N2 cell mucin granules. Cholinergic stimulation of differentiated N2 cell monolayers resulted in depletion of intracellular mucin granules.

The femaleTenebrio molitor genital anlage differentiates from a part of the sternal and abdominal integument of the larva. This epidermal tissue shows a very high regulative capacity during the whole life of the larva. This capacity appears only within the regulative area. The presumptive genital epidermis is composed of labile, morphogenetic fields. It shows however a determined axis of polarity controlled by adjacent abdominal tissues forming a part of the regulative area.

The leg of the last instar larva, and especially the pharate pupa, of Tenebrio molitor (Coleoptera) shows considerable restoration ability. Restoration refers to the regeneration of leg structures removed during the last larval instar. This process involves only those tissues adjacent to the cut surface. At each level of the leg, amputation before a characteristic time leads to the restoration of a complete leg, but thereafter the new segments formed are hypomorphic or just lobes. After an amputation close to the pupal ecdysis, fragments are obtained, and these incomplete structures show that the restoration process has ceased. Just before the pupal ecdysis the leg is withdrawn from the cuticle, so a very late amputation does not effect the leg tissue and a complete pupal and adult leg is formed. The whole of the larval leg participates in adult leg morphogenesis, and histological techniques demonstrate a great correspondence between larval and adult leg segments. The larval trochanter, however, forms the adult trochanter and the proximal part of the femur. The larval tibia forms the adult tibia plus tarsus, except for the adult claws which develop from the larval tarsal claw.

When tissue culture cells were maintained at 37 degrees C in a serum-free medium for 4 hr no change in the number of coated pits could be detected using ultrastructural techniques. However, the number of coated vesicles was highly significantly increased, being 179% more than in the control cultures. If the cells were put back into a medium supplemented with 5% calf serum, the number of coated pits was unchanged, but the number of coated vesicles decreased and returned to the control level within a few minutes. The same results were obtained when using ligands such as Low Density Lipoprotein or alpha-2-macroglobulin which are known to be internalized via coated structures. It is concluded that coated pits appear and disappear at equal rates and that coated vesicles can accumulate independently. It is suggested that this could be due to the presence of a large reserve of soluble clathrin. This pool would have a low turnover rate because cycloheximide did not block coated vesicle accumulation over the period studied.

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an alpha 3 beta 3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets were the tetrameric and dimeric states of the molecule respectively.

Cultured normal and transformed fibroblasts were treated "in situ" by the concanavalin A-peroxidase labelling technique. It is known that peroxidase recognizes only a fraction of the bound lectin depending on the cell type. Kinetics studies revealed that 80 to 95 percent of the peroxidase and only 10 percent of the lectin are released from the cell surface when the labelled cells were reincubated at 37 degrees C. It is shown that it is mostly the concanavalin traced by peroxidase that is released and also that the lectin and the enzyme are shed as a complex or concomitantly. Consequently, the shedding pattern of the enzyme is used to demonstrate heterogeneity in the lectin binding sites; there are two main components labelled by concanavalin and peroxidase, one which has a short period (from 6 to 16 min) and another one with a much longer one (1.3 to 3 h). It is shown that when cells are incubated at 37 degrees C after a lectin treatment, secondary binding forces occur between the lectin and cell surface components which render the lectin unavailable for inhibiting sugars. Under the same conditions, some peroxidase can still be bound and a slight agglutination can still occur.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed and from "normal" hamster cell lines. A glycolipid extract was prepared from these VSV preparations according to the Folch partition procedure. These glycolipids were rendered immunogenic to the Syrian hamsters when incorporated within liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1/1/2 by weight). When the glycolipids were extracted from VSV grown on cell lines (TSV5-cl2 and EHSVi-cl1) which contained the SV40 tumor-specific transplantation antigen (SV40-TSTA), it was possible either to induce a tumor rejection or at least to slow the growth of the tumor in Syrian hamsters challenged with TSV5-cl2 cells. No protection was obtained in animals treated with liposomes containing glycolipids extracted from purified VSV grown on SV40-TSTA-negative cells (EHB). The SV40-TSTA could be a glycolipid of the transformed cell membrane which is incorporated within the VSV envelope.