Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13

Cell growth control mechanisms were studied based on organization of components in glycosphingolipid-enriched microdomain (GEM) in WI38 cells versus their oncogenic transformant VA13 cells. Levels of fibroblast growth factor receptor (FGFR) and cSrc were 4 times and 2-3 times higher, respectively, in VA13 than in WI38 GEM, whereas the level of tetraspanin CD9/CD81 was 3-5 times higher in WI38 than in VA13 GEM. Csk, the physiological inhibitor of cSrc, was present in WI38 but not in VA13 GEM. Functional association of GEM components in control of cell growth in WI38 is indicated by several lines of evidence. (i) Confluent, growth-inhibited WI38 showed a lower degree of FGF-induced MAPK activation than actively growing cells in sparse culture. (ii) The level of inactive cSrc (with Tyr-527 phosphate) was higher in confluent cells than in actively growing cells. Both processes i and ii were inhibited by GM3 since they were enhanced by GM3 depletion with d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). (iii) The high level of inactive cSrc associated with growth-inhibited cells was caused by coexisting Csk in WI38 GEM. (iv) Interaction of GM3 with FGFR was demonstrated by binding of GM3 to FGFR in the GEM fraction, as probed with GM3-coated beads, and by confocal microscopy. In contrast to WI38, both cSrc and MAPK in VA13 were strongly activated regardless of FGF stimulation or GM3 depletion by P4. Continuous, constitutive activation of both cSrc and MAPK was due to (i) a much higher level of cSrc and FGFR in VA13 than in WI38 GEM, (ii) their close association/interaction in VA13 GEM as indicated by clear coimmunoprecipitation between cSrc and FGFR, and (iii) the absence of Csk in VA13 GEM, making GEM incapable of inhibiting cSrc activation.     

Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and mediates binding and internalization of pathogens such as virus (human immunodeficiency virus, hepatitis C), bacteria (Mycobacterium), fungi, and parasites. DC-SIGN expression in vivo is primarily restricted to interstitial dendritic cells (DC) and certain tissue macrophages. We now report that leukemic THP-1 cells, widely used as a model for monocyte-macrophage differentiation, express very low basal levels of DC-SIGN and that DC-SIGN expression in THP-1 cells is regulated during differentiation. Differentiation-inducing agents (phorbol ester, bryostatin) conveyed THP-1 cells with the ability to up-regulate DC-SIGN mRNA levels and cell surface expression in response to interleukin-4 (IL-4) or IL-13. DC-SIGN up-regulation required a functional JAK-STAT signaling pathway, was inhibited in the presence of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha), and conferred THP-1 cells with increased pathogen recognition and T cell stimulatory capabilities. The up-regulation of DC-SIGN on THP-1 cells resembles its inducible expression on monocytes and macrophages, where DC-SIGN expression is also induced by IL-4/IL-13 and negatively regulated by TNF-alpha, LPS, and vitamin D(3). These results point to THP-1 cells as a useful cellular system to characterize the pathogen-binding capabilities of DC-SIGN and to dissect the molecular mechanisms that control its regulated and tissue-specific expression in myeloid dendritic cells, and the results suggest that DC-SIGN constitutes a marker for both DC and alternatively activated macrophages.     

Trehalose maintains phase separation in an air-dried binary lipid mixture

Mixing and thermal behavior of hydrated and air-dried mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-d70-sn-glycero-3-phosphocholine (DSPCd-70) in the absence and presence of trehalose were investigated by Fourier transform infrared spectroscopy. Mixtures of DLPC:DSPCd-70 (1:1) that were air-dried at 25 degrees C show multiple phase transitions and mixed phases in the dry state. After annealing at high temperatures, however, only one transition is seen during cooling scans. When dried in the presence of trehalose, the DLPC component shows two phase transitions at -22 degrees C and 75 degrees C and is not fully solidified at -22 degrees C. The DSPCd-70 component, however, shows a single phase transition at 78 degrees C. The temperatures of these transitions are dramatically reduced after annealing at high temperatures with trehalose. The data suggest that the sugar has a fluidizing effect on the DLPC component during drying and that this effect becomes stronger for both components with heating. Examination of infrared bands arising from the lipid phosphate and sugar hydroxyl groups suggests that the strong effect of trehalose results from direct interactions between lipid headgroups and the sugar and that these interactions become stronger after heating. The findings are discussed in terms of the protective effect of trehalose on dry membranes.     

Metabolism of ovorubin,the major egg lipoprotein from the apple snail

The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad–digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25°C for 12 h with ¹⁴C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad–digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with ³H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.     

Mutagenic stress modulates the dynamics of CTG repeat instability associated with myotonic dystrophy type 1

The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive small pool PCR and sequencing gels. An expansion bias of the stepwise mutation was observed in a (CTG)₁₂₄ allele of a cell line harbouring two modal alleles of 28 and 124 CTG repeats. Interestingly, this expansion bias was clearly enhanced in the presence of mitomycin C. The effect of mitomycin C was also evident in the normal size alleles in two cell lines with alleles of 13/13 and 12/69 repeats, where treated cultures showed new longer alleles. In conclusion, our results indicate that mitomycin C modulates the dynamics of myotonic dystrophy-associated CTG repeats in LBCLs, enhancing the expansion bias of long-pathogenic repeats and promoting the expansion of normal length repeats.     

Effect of cortisol on norepinephrine-mediated contractions in ovine uterine arteries

Cortisol potentiated norepinephrine (NE)-mediated contractions in ovine uterine arteries (UA). We tested the hypothesis that cortisol regulated alpha(1)-adrenoceptor-mediated pharmacomechanical coupling differentially in nonpregnant UA (NUA) and pregnant UA (PUA). Cortisol (10 ng/ml for 24 h) significantly increased contractile coupling efficiency of alpha(1)-adrenoceptors in NUA, but increased alpha(1)-adrenoceptor density in PUA. Cortisol potentiated NE-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] synthesis in both NUA and PUA, but increased coupling efficiency of alpha(1)-adrenoceptors to Ins(1,4,5)P(3) synthesis only in NUA. Carbenoxolone alone did not affect NE-mediated Ins(1,4,5)P(3) production, but significantly enhanced cortisol-mediated potentiation of NE-stimulated Ins(1,4,5)P(3) synthesis in PUA. In addition, cortisol potentiated the NE-induced increase in Ca(2+) concentration in PUA, but increased NE-mediated contraction for a given amount of Ca(2+) concentration in NUA. Collectively, the results indicate that cortisol potentiates NE-mediated contractions differentially in NUA and PUA, i.e., by upregulating alpha(1)-adrenoceptor density leading to increased Ca(2+) mobilization in PUA while increasing alpha(1)-adrenoceptor coupling efficiency and myofilament Ca(2+) sensitivity in NUA. In addition, the results suggest that pregnancy increases type 2 11 beta-hydroxysteroid dehydrogenase activity in the UA.     

Envelope ultrastructure of uncultured naturally occurring magnetotactic cocci

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We studied the surface ultrastructure of uncultured magnetotactic cocci collected from a marine environment by transmission electron microscopy using freeze-fracture and freeze-etching. All bacteria revealed a Gram-negative cell wall. Many bacteria possessed extensive capsular material and a S-layer formed by particles arranged with hexagonal symmetry. No indication of a metal precipitation on the surface of these microorganisms was observed. Numerous membrane vesicles were observed on the surface of the bacteria. Flagella were organized in bundles originated in a depression on the surface of the cells. Occasionally, a close association of the flagella with the magnetosomes that remained attached to the replica was observed. Capsules and S-layers are common structures in magnetotactic cocci from natural sediments and may be involved in inhibition of metal precipitation on the cell surface or indirectly influence magnetotaxis.     

Parasitological analysis of Leonese royalty from Collegiate-Basilica of St. Isidoro, León (Spain): helminths, protozoa, and mites

The royal burial chamber of what is today the Collegiate-Basilica of St. Isidoro in León, Spain, built and remodeled between the 10th and 13th centuries and in the 20th century renamed the Kings' Pantheon, has 13 royal tombs that were opened in the presence of the Abbot-Prior of the Collegiate to enable a group of researchers to obtain all possible information from the royal remains. Several samples were sent to the Parasitology Unit of the Animal Pathology (Animal Health) Department at the Veterinary Faculty of León (Spain). In all the tombs, eggs and remains of nonparasitic mites were observed. In a piece of linen cloth from the bottom of 1 tomb, an Anoplocephala perfoliata egg was found. Furthermore, 4 mummified bodies were found. In 2 of these, those belonging to Infantes María and Fernando, Ascaris lumbricoides eggs were found and in the latter Trichuris trichiura eggs. We have not found in the literature reviewed any records of studies of this kind carried out in Spain.     

Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade

Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.     

Arthropod and filarioid parasites associated with wild rodents in the northeast marshes of Buenos Aires,Argentina

During 1995, 16 species of arthropods and 2 species of filarioids, totaling 1 287 specimens were collected from 64 wild rodents captured in the Hudson Natural Reserve, Buenos Aires, Argentina. Infestation parameters and indexes were analyzed. Host specific richness was S = 6, diversity H = 1.48, and relative density RDI = 40%. High values of parasite species richness and diversity were found on Oligoryzomys delticola (S = 9; H = 1.40), Oxymycterus rufus (S = 9; H = 1.37), and Oligoryzomys flavescens (S = 9; H = 1.28), followed by Scapteromys aquaticus (S = 6; H = 0.17), and Akodon azarae (S = 4; H = 1.20). Deltamys kempi was infested only by Androlaelaps rotundus. O. delticola and O. flavescens showed the highest similarity index (O = 74.19%), followed by O. flavescens with S. aquaticus, as a result of historical processes and shared microhabitats. Considering arthropods-filarioids associations, significant affinity was observed in Litomosoides bonaerensis with Hoplopleura travassosi, Laelaps paulistanensis, and Gigantolaelaps wolffsohni.