Phagosomal Proteins of \u3ci\u3eDictyostelium discoideum\u3c/i\u3e

In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase

A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes

In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small M(r) GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar protons pumps (V-H(+)-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H(+)-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H(+)-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex

Allele-Specific Impairment of GJB2 Expression by GJB6 Deletion del(GJB6-D13S1854)

Mutations in the GJB2 gene, which encodes connexin 26, are a frequent cause of congenital non-syndromic sensorineural hearing loss. Two large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854), which truncate GJB6 (connexin 30), cause hearing loss in individuals homozygous, or compound heterozygous for these deletions or one such deletion and a mutation in GJB2. Recently, we have demonstrated that the del(GJB6-D13S1830) deletion contributes to hearing loss due to an allele-specific lack of GJB2 mRNA expression and not as a result of digenic inheritance, as was postulated earlier. In the current study we investigated the smaller del(GJB6-D13S1854) deletion, which disrupts the expression of GJB2 at the transcriptional level in a manner similar to the more common del(GJB6-D13S1830) deletion. Interestingly, in the presence of this deletion, GJB2 expression remains minimally but reproducibly present. The relative allele-specific expression of GJB2 was assessed by reverse-transcriptase PCR and restriction digestions in three probands who were compound heterozygous for a GJB2 mutation and del(GJB6-D13S1854). Each individual carried a different sequence variant in GJB2. All three individuals expressed the mutated GJB2 allele in trans with del(GJB6-D13S1854), but expression of the GJB2 allele in cis with the deletion was almost absent. Our study clearly corroborates the hypothesis that the del(GJB6-D13S1854), similar to the larger and more common del(GJB6-D13S1830), removes (a) putative cis-regulatory element(s) upstream of GJB6 and narrows down the region of location

Patterns of Spiny Lobster (Panulirus argus) Postlarval Recruitment in the Carribbean: A CRTR Project

As part of the Coral Reef Targeted Research (CRTR) Program, a partnership between the Global Environment Facility and the World Bank, our research team examined the recruitment patterns of Caribbean spiny lobster (Panulirus argus) postlarvae among regions in the Caribbean, with a particular focus on Mesoamerica. Our goal was to collect comparable information on postlarval supply among regions and to provide data to test predictions of connectivity generated from a coupled biophysical oceanographic model of lobster larval dispersal. Here we present the results of the postlarval recruitment monitoring program. We monitored the catch of postlarvae on Witham-style collectors at sites in the Caribbean from March 2006 to May 2009, although the duration and frequency of sampling varied among locations. Recruitment varied considerably among months and locations. It peaked in the Western Caribbean in the fall (Oct - Dec), whereas in Florida, Puerto Rico, and Venezuela peaks were in spring (Feb - April) with a smaller peak in the fall. Sites generally fell into two groups with respect to monthly variability in recruitment: low variability sites (e.g., Honduras, southern Mexico, Venezuela) and high variability sites (e.g., Florida, San Andres Islands, Puerto Rico, northern Mexico). Recruitment magnitude varied locally, but generally increased (lowest to highest) from Puerto Rico, San Andres Islands, Honduras, Mexico, Venezuela, to Florida. Recruitment trends mirrored fishery catch in some locations, implying a recruit-to-stock linkage. Recruitment was significantly correlated among several sites, suggesting similarity in their larval sources and oceanographic regimes

Mega-tsunami conglomerates and flank collapses of ocean island volcanoes

Co-auteur étrangerInternational audienceMarine conglomerates at high elevation on the flanks of ocean islands are usually interpreted as evidence of mega-tsunamis generated by volcano flank collapses, although their origin is sometimes debated (elevated littorals vs. tsunami). In this review, we introduce case studies of well-documented examples of tsunami conglomerates in Hawaii (Pacific Ocean), the Canary and Cape Verde Islands (Atlantic Ocean), and Mauritius Island (Indian Ocean). Other less-documented marine conglomerates are also presented as tsunami candidates. Then, we build a comprehensive picture of the general characteristics of these conglomerates and the different methods that can be applied to date them. Different perspectives of research are proposed, especially on the use of tsunami conglomerates as proxies for better constraining numerical models of ocean island flank collapses and associated tsunamis. We also discuss the possible links between volcano growth, flank instability, and climate

Genotyping with a 198 Mutation Arrayed Primer Extension Array for Hereditary Hearing Loss: Assessment of Its Diagnostic Value for Medical Practice

Molecular diagnostic testing of individuals with congenital sensorineural hearing loss typically begins with DNA sequencing of the GJB2 gene. If the cause of the hearing loss is not identified in GJB2, additional testing can be ordered. However, the step-wise analysis of several genes often results in a protracted diagnostic process. The more comprehensive Hereditary Hearing Loss Arrayed Primer Extension microarray enables analysis of 198 mutations across eight genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, MTRNR1 and MTTS1) in a single test. To evaluate the added diagnostic value of this microarray for our ethnically diverse patient population, we tested 144 individuals with congenital sensorineural hearing loss who were negative for biallelic GJB2 or GJB6 mutations. The array successfully detected all GJB2 changes previously identified in the study group, confirming excellent assay performance. Additional mutations were identified in the SLC26A4, SLC26A5 and MTRNR1 genes of 12/144 individuals (8.3%), four of whom (2.8%) had genotypes consistent with pathogenicity. These results suggest that the current format of this microarray falls short of adding diagnostic value beyond the customary testing of GJB2, perhaps reflecting the array's limitations on the number of mutations included for each gene, but more likely resulting from unknown genetic contributors to this phenotype. We conclude that mutations in other hearing loss associated genes should be incorporated in the array as knowledge of the etiology of hearing loss evolves. Such future modification of the flexible configuration of the Hereditary Hearing Loss Arrayed Primer Extension microarray would improve its impact as a diagnostic tool

Avance en el diseño de un péptido bloqueador del receptor opioide kappa 2 humano

Se evaluó la posibilidad de predecir una probable estructura secundaria para el Receptor Opioide Kappa 2 humano tomando como base la secuencia de aminoácidos del Receptor Opioide Kappa 1 humano. La estructura predicha mostró ser compatible con los datos que se poseen acerca de este tipo de receptores. Con esta prueba inicial, el proyecto que tiene como objetivo principal diseñar un análogo proteico para el Receptor Opioide Kappa 2 humano, ha mostrado el nivel mínimo de viabilidad necesario para ser continuado

Post-Operative Functional Outcomes in Early Age Onset Rectal Cancer

Background: Impairment of bowel, urogenital and fertility-related function in patients treated for rectal cancer is common. While the rate of rectal cancer in the young (<50 years) is rising, there is little data on functional outcomes in this group. Methods: The REACCT international collaborative database was reviewed and data on eligible patients analysed. Inclusion criteria comprised patients with a histologically confirmed rectal cancer, <50 years of age at time of diagnosis and with documented follow-up including functional outcomes. Results: A total of 1428 (n=1428) patients met the eligibility criteria and were included in the final analysis. Metastatic disease was present at diagnosis in 13%. Of these, 40% received neoadjuvant therapy and 50% adjuvant chemotherapy. The incidence of post-operative major morbidity was 10%. A defunctioning stoma was placed for 621 patients (43%); 534 of these proceeded to elective restoration of bowel continuity. The median follow-up time was 42 months. Of this cohort, a total of 415 (29%) reported persistent impairment of functional outcomes, the most frequent of which was bowel dysfunction (16%), followed by bladder dysfunction (7%), sexual dysfunction (4.5%) and infertility (1%). Conclusion: A substantial proportion of patients with early-onset rectal cancer who undergo surgery report persistent impairment of functional status. Patients should be involved in the discussion regarding their treatment options and potential impact on quality of life. Functional outcomes should be routinely recorded as part of follow up alongside oncological parameters