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.     

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.     

Rock phosphate solubilization with gluconic acid produced by immobilized Penicillium variabile P16

Summary Penicillium variabile P16 immobilized on polyurethane sponge produced gluconic acid in presence of rock phosphate, the latter being simultaneously solubilized during five repeated batches. A total production of 42, 60, and 90 g gluconic acid/l was obtained for 3, 7, and 14 g rock phosphate/l, respectively. Accordingly, soluble phosphorus concentration increased with gluconic acid production, reaching a maximum of 350 mg/l at the 3d batch in medium supplemented with 14 g rock phosphate/l.     

Effect of CMV-immunoglobulins (cytotect biotest) prophylaxis on CMV pneumonia after lung transplantation

Lung transplant (LT) recipients among solid organ transplant recipients are at high risk for cytomegalovirus (CMV) infections. We evaluated the effect of CMV-Immunoglobulins (CMV-IG) (Cytotect Biotest) on CMV pneumonia diagnosed in 303 follow-up transbronchial biopsies (TBB) of lung transplant recipients. 24 patients (control group, 155 TBB from 1999 to 2002) received acyclovir for 24 months and 33 recipients (study group, 148 TBB from 2003 to 2008) received a combined CMV prophylaxis consisting of CMV-IG (Cytotect Biotest) for 12 months and a short Ganciclovir or Valganciclovir therapy from 21th to 42th postoperative day followed by acyclovir up to 24 months. In our study the percentage of pneumonia at first month TBB was similar in the study group vs the control group, 9.1% (3/33) vs 8.3% (2/24), p=0.9 ns, but after the first month the percentage was significantly lower in the study group in the first year at follow-up TBB, 1% (1/99) vs 6.4% (5/78), p=0.048, and in first two years follow-up TBB, 0.8% (1/122) vs 6.5% 8/124), p=0.018 (Statistical analysis: Chi-square test for proportion differences). Our data suggest a strong efficacy of CMV-IG prophylaxis in reducing CMV pneumonia after first month in lung transplant recipients.     

Organization and possible origin of the Bari-1 cluster in the heterochromatic h39 region of Drosophila melanogaster

The molecular organization of the heterochromatic h39 region of the Drosophila melanogaster second chromosome has been investigated by studying two BAC clones identified both by Southern blotting and by FISH experiments as containing tandem arrays of Bari1, a transposable element present only in this region. Such BAC clones appear to contain different portions of the h39 region since they differ in the DNA sequences flanking the Bari1 repeats on both sides. Thus, the 80 Bari1 copies estimated to be present in the h39 region are split into at least two separated subregions. On the basis of the analysis of the flanking sequences a possible mechanism depending on an aberrant activity of the Bari1 transposase is proposed for the genesis of the heterochromatic tandem arrays of the element.     

Systematic consideration of floral microcharacters of the South American genus Chrysolaena (Vernonieae,Asteraceae)

Floral microcharacters of the genus Chrysolaena H. Rob. (Vernonieae, Asteraceae) were analysed in detail for the first time in order to evaluate the taxonomic position of conflictive species in the group. The results were also compared with studies carried out in species of related genera. In addition to distinctive microcharacters previously studied in some species of the genus, other characters such as trichome types of the corolla, style, anthers and cypselae have been analysed for the first time. The presence of glandular apical appendage and cypselae are common characteristics among species Chrysolaena. In addition to these, this study shows that presence of glands on the style and corollas is another consistent characteristic in the genus. However, the absence of basal stylar node would not be a diagnostic character since this varies widely among species. The results indicate that most of the microcharacters of Chrysolaena analysed are quite consistent in the genus, but they are no more consistent than the pollen morphology (type 'C') and chromosome base number (x = 10). Until now, these last two features would be most useful for separating Chrysolaena from the related genera Lessingianthus and Lepidaploa. At species level, the results show that related species can be distinguished by the different combinations of floral microcharacters. The value of microcharacters could be increased if they are combined with other morphological, cytological, and palynological data.