Covalent cross-linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29)

[125I]Monoiodinated vasoactive intestinal peptide (125I-VIP) was cross-linked with human colonic adenocarcinoma cells (HT29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A major polypeptide of Mr = 67 000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of 125I-VIP covalent cross-linking with this polypeptide was very similar to that giving half-maximum displacement of 125I-VIP on HT 29 cells (0.6 nM). Glucagon or insulin was unable to inhibit the labelling of the Mr-67 000 component. In our experimental conditions neither specific 125I-VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor. Preincubation of HT 29 cells with native VIP at 37 degrees C, before 125I-VIP binding and subsequent cross-linking reaction, decreased the labelling of the Mr-67 000 polypeptide up to 80%. Assuming one molecule of 125I-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr = 64 000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.     

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.     

Polarized distribution of γ interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells)

Summary The fine structure of single identified muscle fibers and their nerve terminals in the limb closer muscle of the shore crab Eriphia spinifrons was examined, using a previous classification based on histochemical evidence which recognizes a slow (Type-I) fiber and three fast (Type-II, Type-III, Type-IV) fibers. All four fiber types have a fine structure characteristic of crustacean slow muscle, with 10–12 thin filaments surrounding each thick filament and sarcomere lengths of 6–13 m. Type-IV fibers have sarcomere lengths of 6 m while the other three types have substantially longer sarcomeres (10–13 m). Structural features of nerve terminals revealed excitatory innervation in all four fiber types but inhibitory innervation in Type-I, Type-II, and Type-III fibers only. Thus fibers with longer sarcomeres receive the inhibitor axon but those with shorter sarcomeres do not. Amongst the former, synaptic contact from an inhibitory nerve terminal onto an excitatory one, denoting presynaptic inhibition, was seen in Type-I and Type-II fibers but not in Type-III and Type-IV fibers. Inhibitory innervation of the walking leg closer muscle is therefore highly differentiated: some fibers lack inhibitory nerve terminals, some possess postsynaptic inhibition, and some possess both postsynaptic and presynaptic inhibition.     

Short-term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29-D4 cells.

Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation.     

Evidence for an ectophosvitin kinase activity that phosphorylates a 123 kDa endogenous substrate on a human colonic adenocarcinoma cell (HT 29)

When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kinase activities. We have characterized one of them as an ectophosvitin kinase which phosphorylated specifically a 123 kDa polypeptide and whose expression or accessibility varied according to cell density.     

Efficacy of curcumin in the treatment of experimental vulvovaginal candidiasis

BackgroundCandida albicans is the main agent that causes vulvovaginal candidiasis. Resistance among isolates to azole antifungal agents has been reported.AimsDue to the well-known antifungal potential of curcumin, the purpose of this work was to evaluate the in vitro anticandidal activity of curcumin and its effect in the treatment of experimental vulvovaginal candidiasis.MethodsThe anticandidal activity of curcumin was investigated against eight Candida strains by the broth microdilution assay, and its mechanism of action was evaluated by testing the binding to ergosterol. Then, the effect of curcumin in the treatment of vulvovaginal candidiasis was evaluated in an immunosuppressed, estrogen treated rat model.ResultsCurcumin showed minimum inhibitory concentration values of 125–1000 μg/ml, and the best result was observed against Candida glabrata. The compound was shown to be able to bind to the ergosterol present in the membrane, event that may be the mechanism of action. In addition, in the in vivo model of vulvovaginal candidiasis with C. albicans, treatments reduced the vaginal fungal burden in infected rats after seven days of treatment with different doses.ConclusionsCurcumin could be considered a promising effective antifungal agent in the treatment of vulvovaginal candidiasis.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

The concept of specificity and the Italian contribution to the discovery of the malaria transmission cycle

At the dawn of the 20th century, the change in the scientific, political and cultural attitudes towards malaria was the result of the discovery of the theoretical simplicity of the malaria transmission cycle and of the possibility to interrupt it, by avoiding the contacts between people and mosquitoes. The 'mosquito hypothesis', suggested already in the 1880s, had to be included into a coherent scientific theory, in which a fundamental part was played by the concept of specificity. The paper analyses the Italian contribution to this scientific change and the epistemological aspects of the debate between Ronald Ross and Battista Grassi about their respective role in the discovery of the human malaria transmission cycle. This debate has been often interpreted in sociological or psychological terms. However, behind the dispute there is a different definition of what is a scientific explanation in biological sciences and in particular in parasitology. This point is made clear by the analysis of four different theoretical problems implied in the discovery of the transmission cycle: the concept of specificity, the comparative method in parasitology, the specificity of the life-cycle of parasites and vectors, and the role of the analogical reasoning in science and medicine.     

Orientation at night: an innate moon compass in sandhoppers (Amphipoda: Talitridae)

The supralittoral amphipod Talitrus saltator is well known for its capacity for astronomical orientation using the sun and moon as compasses. It has also been demonstrated that the sun compass is innate in this species. In our experiments, we released inexpert (naive) young born in the laboratory into a confined environment under the full moon and in the absence of the horizontal component of the magnetic field. They were allowed to see the natural sky and the moon only at the moment of release. The young individuals were obtained in the laboratory by crossing adult individuals from the same and different populations of sandhoppers. The young from intrapopulation crosses were well oriented towards the directions corresponding to those of their parents, whereas the young from interpopulation crosses were oriented in an intermediate direction. Therefore, our experiments demonstrate in the sandhopper T. saltator that sea-land moon orientation relies on an innate chronometrically compensated mechanism.