Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.     

Phylogenetic relationship among all living species of the genusBubalus based on DNA sequences of the cytochromeb gene

The cytochromeb genes of all living species ofBubalus, including the river type and the swamp type of domestic buffaloes (Bubalus bubalis), were sequenced to clarify their phylogenetic relationships. These sequences were compared together with the African buffalo (Syncerus caffer) and banteng (Bos javanicus) sequences as an outgroup. Phylogenetic trees ofBubalus species based on the DNA sequences of the cytochromeb gene demonstrated that the tamaraw (Bubalus mindorensis), endemic to the Philippines, could be classified into the subgenusBubalus, not the subgenusAnoa. The divergence time between the lowland anoa (B. depressicornis) and the mountain anoa (B. quarlesi) was estimated at approximately 2.0 million years (Myr), which is almost the same as the coalescence time for theBubalus sequences. This large genetic distance supports the idea that the lowland anoa and the mountain anoa are different species. An unexpectedly large genetic distance between the river and the swamp type of domestic buffaloes suggests a divergence time of about 1.7 Myr, while the swamp type was noticed to have the closest relationship with the tamaraw (1.5 Myr). This result implies that the two types of domestic buffaloes have differentiated at the full species level.     

Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin-disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the beta-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the beta-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.     

Entamoeba histolytica: microtubule movement during mitosis

The movement of microtubules (MTs) during nuclear division of Entamoeba histolytica was ultrastructurally studied. Regarding this MT movement, five stages of mitosis could be defined: prophase, metaphase, anaphase A, anaphase B, and telophase. In early stages of mitosis, chromatinic material appeared condensed, and MTs were detected in the center of the nucleus. Later, MTs seemed to grow from an electron-dense body located in the center of the nucleus. This body might be the microtubule organizing center, which organized the MTs, first in a lateral way, and later to form the mitotic spindle, which was made of a bundle of MTs joined by their ends. This junction of MTs to themselves could also be observed in cross-sections. The last stage of mitosis was the nuclear separation. Two different morphological types of intranuclear vesicles were also observed, which seemed to have different types of membrane. Both intranuclear vesicles were present during nuclear division, generally in clusters, and located close to the nuclear periphery.     

Flavonol glycosides from Monnina sylvatica.

A new kaempferol triglycoside and three known kaempferol glycosides, among them two apiosides, have been isolated from the aerial parts of Monnina sylvatica. The structures were established on the basis of acid and enzymatic hydrolysis and spectral data (UV, 1H and 13CNMR, NOE difference measurements, D/CI and FAB-MS) of the isolates and of some derivatives. The triglycoside kaempferol 3-O-beta-D-glucosyl-(1----2)-O-[alpha-L-rhamnosyl(1----6)]-beta-D- galactoside is a new natural product. The configuration of the apiosyl moiety in kaempferol 3-O-beta-D-apiosyl(1----2)-beta-D-galactoside and kaempferol 3-O-beta-D-apiosyl(1----2)-O-[alpha-L-rhamnosyl(1----6)]- beta-D-galactoside was established through NOE difference measurements on the peracetate.     

Electronic particle size measurements of platelet aggregates formed in vitro.

The aggregation of human platelets induced by adenosine diphosphate (ADP) was used to evaluate electronic particle size analyzer measurements of platelet aggregates in plasma. As platelets began to clump in plasma, the total volume and the diameter of individual aggregates increased; after a time dependent on experimental conditions, the diameter increased but the total volume remained unchanged. Similar but opposite changes in size distribution occurred during platelet deaggregation. The total volume of aggregates formed in plasma varied (linear correlation coefficient = 0.99) with the total volume of platelets which were available to clump and with simultaneous changes in optical density. The diameter of the aggregates varied with the concentration of, and time of exposure to, ADP and with the total volume of platelets and aggregates in plasma was not different from that of control platelets in untreated plasma, the individual platelets aggregated without an accompanying increase in size. This study demonstrates that platelet aggregation can be characterized by electronic measurements of the size distribution of platelet aggregates.     

Contribution of Bioactive Compounds to the Antioxidant Capacity of the Edible Mushroom Neolentinus lepideus

Neolentinus lepideus is a fungus consumed by rural communities in Central America and Asia due to its rich flavor; however, little information on its chemical composition is available. With this in mind, the objective of this work was to determine the content of vitamin E and C, ergosterol, and phenolic compounds of this fungus, as well as its antioxidant capacity. The quantified bioactive compounds were two isoforms of vitamin E, highlighting α-tocopherol (3370.35 mg/100 g dry weight, DW) and ergosterol (11.70 mg/100 g DW). The total phenolic content was 164.80 mg gallic acid equivalents/100 g, and nine phenolic compounds were identified (protocatechuic, p-hydroxybenzoic, caffeic, vanillic, ferulic, salicylic, p-anisic, trans-cinnamic acids, and scopoletin). The highest antioxidant capacity was detected in the lipophilic extract with TEAC (27688 μmoles Trolox equivalents/100 g). These results suggest that lipophilic compounds are among the main bioactive compounds in N. lepideus, and they might exhibit the highest radical scavenging properties in non-polar extracts.     

Recovery of Motor Deficit,Cerebellar Serotonin and Lipid Peroxidation Levels in the Cortex of Injured Rats

The sensorimotor cortex and the cerebellum are interconnected by the corticopontocerebellar (CPC) pathway and by neuronal groups such as the serotonergic system. Our aims were to determine the levels of cerebellar serotonin (5-HT) and lipid peroxidation (LP) after cortical iron injection and to analyze the motor function produced by the injury. Rats were divided into the following three groups: control, injured and recovering. Motor function was evaluated using the beam-walking test as an assessment of overall locomotor function and the footprint test as an assessment of gait. We also determined the levels of 5-HT and LP two and twenty days post-lesion. We found an increase in cerebellar 5-HT and a concomitant increase in LP in the pons and cerebellum of injured rats, which correlated with their motor deficits. Recovering rats showed normal 5-HT and LP levels. The increase of 5-HT in injured rats could be a result of serotonergic axonal injury after cortical iron injection. The LP and motor deficits could be due to impairments in neuronal connectivity affecting the corticospinal and CPC tracts and dysmetric stride could be indicative of an ataxic gait that involves the cerebellum.