Specific Developmental Defects in Molluscs after Treatment with Retinoic Acid during Gastrulation

All-trans retinoic acid is known as a teratogen in vertebrate development. To study whether molluscan morphogenesis is sensitive to retinoic acid, the development of Lymnaea stagnalis, Physa fontinalis and Bithynia tentaculata was examined after treatment with retinoic acid. Low concentrations retinoic acid (10^–7M) specifically affected eye formation in each of these species. In Lymnaea , it was shown that 10^–6M retinoic acid resulted in a wider spectrum of deficiencies, including eye defects, arrested development and shell deformations. Pulse treatments revealed that embryos were most sensitive during gastrulation. Soon after gastrulation the embryos had lost their sensitivity to retinoic acid, which indicates that the observed defects do not result from a general non-specific toxic effect on cells. Even though molluscan development differs in numerous respects from vertebrate development, the present results suggest that molluscs share common features with vertebrates in morphogenetic processes that operate in early development.     

The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T. neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation.     

Ustilago maydis Mating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bolker, M., and Kahmann, R. 1996. Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genetics and Biology 20, 299-312. When small drops of Ustilago maydis sporidia were placed 100-200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example, a2b2 mating hyphae grew toward a1b1 and a1b2 mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with the a2 allele began elongating before sporidia with the a1 allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous at b but not at a formed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, the a2a2b1b2 strain formed filaments more quickly than the a1a1b1b2 strain. Taken together, these results suggest that the a2 pheromone diffuses less readily or is degraded more quickly than the a1 pheromone.     

Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus,Gastropoda)

Summary The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3–1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5–50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100–200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs — as in untreated eggs — microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.     

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.     

SUMO protein modification

SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanistically related to ubiquitin in that they are posttranslationally attached to other proteins. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds formed between its C-terminal glycine residue and the epsilon-amino group of internal lysine residues. The enzymes involved in the reversible conjugation of SUMO are similar to those mediating the ubiquitin conjugation. Since its discovery in 1996, SUMO has received a high degree of attention because of its intriguing and essential functions, and because its substrates include a variety of biomedically important proteins such as tumor suppressor p53, c-jun, PML and huntingtin. SUMO modification appears to play important roles in diverse processes such as chromosome segregation and cell division, DNA replication and repair, nuclear protein import, protein targeting to and formation of certain subnuclear structures, and the regulation of a variety of processes including the inflammatory response in mammals and the regulation of flowering time in plants.     

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.     

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.     

Nouveaux sites paléolithiques anciens en République de Djibouti : bilan préliminaire de prospections récentes dans le Bassin du Gobaad,Afar central

New sites from the Lower Paleolithic of the Republic of Djibouti: Initial results from a recent survey of the Gobaad Basin, Central Afar. Previous research in the Republic of Djibouti resulted in two notable Paleolithic findings: the Oldowan elephant butchery site of Barogali, excavated by J. Chavaillon and A. Berthelet, and a Homo erectus/sapiens maxilla described by L. de Bonis et al. These discoveries were made in the 1980s, and no paleoanthropological surveys have been conducted in Djibouti in the following decades. In 2007, the Mission archéologique et paléontologique Afar Djibouti (MAPAD) carried out a new survey of the Gobaad Basin and discovered several new archaeological and paleontological sites attributed to the Lower Paleolithic. Three sites in particular contain rich concentrations of lithic artifacts on the surface that, based on field examination, can be attributed to the Oldowan. Of these, the site of Chekheyti Issie 3 (CKI-3) is the largest, comprising a surface of well over 100 m² of abundant Oldowan lithics in spatial association with fossil hippopotamus remains. The presence of lithic refits, identified in an ad hoc fashion in the field, suggests that the site was minimally disturbed. Further excavation and analysis of CKI-3 should provide insight into carcass acquisition and processing by early hominids. More generally, the newly discovered sites in the Gobaad Basin will allow for the testing of a range of hypotheses regarding both local and regional variation in hominid technology, behavior, and subsistence strategies in the Lower Pleistocene.