Endocytosis and signaling: a relationship under development

The ability to internalize macromolecules by endocytosis is a property of all eukaryotic cells. Frontline research on endocytosis has been presented in a successful series of biannual meetings in Europe. This year's meeting on "Membrane Dynamics in Endocytosis" was held September 13-18 in Acquafredda di Maratea, on the coast of southern Italy. Four key questions were addressed: What are the molecular mechanisms of endocytic membrane trafficking? How does endocytosis modulate receptor signaling and vice versa? What is the importance of endocytosis during development? How do endocytic organelles contribute to immunity or susceptibility to pathogens?     

Production of alkaloids by in vitro culture of Erythrina americana Miller

The production of erythroidines and other alkaloids was studied in cotyledons, callus and cell suspension cultures of Erythrina americana Miller. The cell suspension cultures, grown in Murashige & Skoog medium with naphthaleneacetic acid (3 mg l^–1) and kinetin (2 mg l^–1), produced 89 and 17 g - and -erythroidines respectively per g dry wt.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Genotypic fidelity of micropropagated pineapple (Ananas comosus) plantlets assessed by isozyme and RAPD markers

In the present work, pineapple plantlets (cv. `Amarelinho') micropropagated by stationary (S) and temporary immersion (T) systems were evaluated in terms of genotypic fidelity by isozyme and RAPD markers. Neither isozymes (average 0.67%) nor RAPDs (average 7.5%) alone detected significant differences between the two micropropagated systems. However, when combined isozymes and RAPDs data more somaclonal variants were detected in S than T, with RAPDs revealing more variation than isozymes. In the T system, the treatment with PBZ 6.0 M gave the greatest occurrence of variants (8.9%), although significant statistical differences (; p>0.05) between presence and absence of PBZ or GA were not detected. Temporary immersion in comparison with the stationary system resulted in the lowest proportion of somaclonal variants (1.9% vs. 3.9%). Although it does not represent an in depth genome analysis, ours is the first work that has attempted to evaluate the genotypic fidelity of micropropagated pineapple plantlets.     

Iridoid glucosides from Randia spinosa (Rubiaceae)

An iridoid glucoside: randinoside, along with five known iridoids: galioside, deacetylasperulosidic acid methyl ester, scandoside methyl ester, geniposide and gardenoside, were isolated from the stems of Randia spinosa. The structures were determined by spectroscopic analysis, including 2D NMR techniques.     

A reduced protein model with accurate native-structure identification ability

A protein model that is simple enough to be used in protein-folding simulations but accurate enough to identify a protein native fold is described. Its geometry consists of describing the residues by one, two, or three pseudoatoms, depending on the residue size. Its energy is given by a pairwise, knowledge-based potential obtained for all the pseudoatoms as a function of their relative distance. The pseudoatomic potential is also a function of the primary chain separation and residue order. The model is tested by gapless threading on a large, representative set of known protein and decoy structures obtained from the "Decoys 'R' Us" database. It is also tested by threading on gapped decoys generated for proteins with many homologs. The gapless threading tests show near 98% native-structure recognition as the lowest energy structure and almost 100% as one of the three lowest energy structures for over 2200 test proteins. In decoy threading tests, the model recognized the majority of the native structures. It is also able to recognize native structures among gapped decoys, in spite of close structural similarities. The results indicate that the pseudoatomic model has native recognition ability similar to comparable atomic-based models but much better than equivalent residue-based models.     

Endocytic trafficking during Drosophila development

During the last decade, many of the factors and mechanisms controlling membrane and protein trafficking in general and endocytic trafficking in particular have been uncovered. We have a detailed understanding of the different endocytic trafficking steps: plasma membrane budding, endocytic vesicle motility and fusion with the endosome, recycling, transcytosis and lysosomal degradation. The kinetics and trafficking pathway of many signaling receptors and the relevance of endocytic trafficking during signaling in many mammalian cultured cells are also well understood. However, only in recent years has the role of endocytic trafficking during cell-to-cell communication during development, i.e. during patterning, induction and lateral inhibition, begun to be explored. The contribution of Drosophila developmental genetics and cell biology has been fundamental in elucidating the essential role of endocytosis during these processes. Reviewed here are some of the recent developments on the role of endocytic trafficking during long- and short-range signaling and during lateral inhibition.     

Purification and characterization of a lectin from Annona coriacea seeds

A novel lectin, denominated ACLEC, was isolated from Annona coriacea seeds, belonging to the Annonaceae family. The lectin presented one protein band in SDS-PAGE of 14 kDa. Of the sugars tested, Dglucose and D-mannose were the best inhibitors. A search sequence database showed that ACLEC had homology with other plant lectins, belonging to leguminous lectin family.     

A novel function for Hedgehog signalling in retinal pigment epithelium differentiation

Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.