Occurrence of Cu-ATPase in Dictyostelium: possible role in resistance to copper

Dictyostelium discoideum amoebae showed an uncommon resistance to Cu(2+), as pointed out through cell growth rate (EC(50) = 469 +/- 30 microM) and the neutral red cytotoxicity assay (EC(50) = 334 +/- 45 microM). Although no evidence of Cu-inducible metallothionein was found, Cu-dependent ATPase activity was cytochemically detected on pelletted, resin-embedded amoebae. This activity required Cu(2+) in the incubation medium, was sensitive to TPEN, vanadate and temperature, and showed dose-dependent increase after exposure of amoebae to 10-500 microM Cu(2+) for 7 days. Accordingly, immunofluorescence and Western blotting revealed the occurrence of a Cu-inducible, putative homologue of human Menkes (MNK) Cu-P-type ATPase. To verify if Cu-ATPase is involved in copper resistance, amoebae were exposed to low concentrations of Cu(2+) and vanadate followed by the neutral red assay. Exposure to either treatment showed no effect, while a combination caused a dramatic increase of Cu toxicity, possibly depending on Cu-ATPase inhibition.     

Evaluation of the impact of recreational dive activity on the community structure of some coral reefs at Los Roques Archipelago National Park, Venezula

In order to evaluate if snorkeling had significant effects on coral community structure, three different coral reefs (Madrizquí, Pelona de Rabusquí and Crasquí) located at Archipelago Los Roques National Park, Venezuela, were surveyed. For each site, the coral community structure of two different areas, one subjected to intense snorkeling use (FB) and other not frequently used (PFB), were compared. Community structure was determined with 1 m2-quadrants and 20 m-long transects. These communities were described in terms of species richness, diversity (Shannon-Wiener) and evenness indexes, live and dead coral cover and cover of other organisms (sponges, octocorals and algae). Comparisons within sites were performed with a Kruskall-Wallis test. A total of 24 species of scleractinian corals were found. Live coral cover ranged from 29.9% +/- 26.43 (Crasquí) to 34.55% +/- 6.43 (Madrizquí), while dead coral cover ranged from 32.51% +/- 2.86 (Madrizquí) to 60.78% +/- 21.3 (Pelona de Rabusquí). The PFB areas showed higher live coral cover compared to FB areas; however, significant differences were only found in Crasquí and Pelona de Rabusquí (p < 0.05). Species richness, diversity and evenness were variable and no trends were observed between FB and PFB areas. The frequency of both damaged and diseased colonies were low (< 1%), most damages observed were natural (parrotfish predation). Damages caused by divers such as fin impacts, were not found at the reefs studied. These results suggest that, currently, diving pressure is not as high to cause massive loses of live coral cover in these reefs. However, the lack of strict controls for these activities might produce long-term changes in the structure of these coral communities.     

Present state of diseases and other signs of reef deterioration at seven coral reefs at Los Roques Archipelago National Park, Venezuela

This work was aimed to determine the incidence of coral diseases in six different reef sites at the Parque Nacional Archipiélago de Los Roques, Venezuela: Arrecife de herradura, Arrecife costanero, both at Dos Mosquises Sur Key, Boca de Cote, Carenero, Crasquí and Pelona de Rabusquí. Each reef was surveyed by using ten 10 m2-band transects (10 x 1 m), placed parallel to the long axis of the reef within a depth gradient ranging from 1 to 9 m depth. All healthy and injured corals, along each band transect, were counted and identified to species level. Additionally, all diseases and recent mortality that were still identifiable on each colony were also recorded. The occurrence of diseased colonies and other signs of reef decline between localities were compared by means of a Chi2 test. The absolute, relative and mean incidence was estimated for each disease and other signs of damage observed for all coral species surveyed at each site. The overall incidence of coral diseases was low for all the localities surveyed, only 6.04% of the 3 344 colonies observed, showed signs of diseases. The most important diseases recorded were the Yellow-Blotch Disease (YBD) and Dark Spots Disease (DSD) with 2.1% +/- 1.52 y 2.1% +/- 2.54, respectively. Significant differences were found in the incidence of coral diseases between reef sites (Chi2 p < 0.05). Finally, the occurrence of colonies injured by parrotfish bites and pomacentrids was higher compared with the incidence of coral diseases for all the reefs surveyed. In conclusion, currently the proportion of healthy colonies at Los Roques coral reefs is higher than the percentage of both diseased and injured colonies.     

Disease in scleractinian corals: a new problem in the reef at Cayo Sombrero, Morrocoy National Park, Venezuela?

At the beginning of 1996 coral reefs in Morrocoy National Park, Venezuela, suffered an unprecedented mass mortality event. As a consequence, live coral cover dropped to 2-10%. One of the few reefs that kept live coral cover over 35% was Cayo Sombrero; nonetheless, the presence of some coral diseases has been detected within the past 2 years, representing a new source of coral mortality. Due to this situation, this study started a monitoring program on the incidence of coral diseases and syndromes in the reef of Cayo Sombrero. The CARICOMP protocol was used in order to evaluate reef health. Ten parallel band-transects (20 x 2m) where established at two depth intervals: Five between 3-8 m and five between 8-12 m, and the frequency of both, healthy and unhealthy colonies of each coral species was recorded along each band transect. In addition to other sources of coral damage (predation, siltation, etc), significant differences in disease incidence between the two depths intervals were tested with a Kruskall-Wallis test. The main problems observed were coral diseases such as yellow band (4.2%), dark spots (1.61%) and white plague-II (1.4%), mainly affecting Montastraea faveolata, M. annularis and Siderastrea siderea. Siltation, affecting massive colonies, such as Colpophyllia natans and Diploria strigosa; algae overgrowth, predation, anchor damage, and bleaching. Significant differences were found in the incidence of unhealthy (Kruskall-Wallis, p < 0.05) bleached (Kruskall-Wallis, p < 0.05) and colonies affected by siltation (Kruskall-Wallis, p < 0.05). More than 60% of the 585 coral colonies surveyed at both depths were found to be healthy, indicating that the Cayo Sombrero reef is still in good conditions compared to other localities in the Park. This study stresses the need to conduct early monitoring programs that survey coral disease incidence as a source of mortality for this coral reef.     

Effect of a modified thymine on the structure and stability of [d(TGGGT)]4 quadruplex

Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their stability is ion-dependent. In this work we compare the stability of [d(TGGGT)]₄ and [d(T*GGGT)]₄ quadruplexes. The last one contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the [d(TGGGT)]₄ sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the G-quadruplex formation. NMR and CD spectra of [d(T*GGGT)]₄ were characteristic of parallel-stranded, tetramolecular quadruplex. CD and DSC melting experiments reveal that [d(T*GGGT)]₄ is less stable that unmodified quadruplex. Molecular models suggest possible explanation for the observed behaviour.     

Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice

Small animal models with the capacity to support engraftment of a functional human immune system are needed to facilitate studies of human alloimmunity. In the present investigation, non-obese diabetic (NOD) severe combined immunodeficient (scid) beta2-microglobulin-null (B2mnull) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2mnull mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2mnull mice were established by injection of human spleen cells or PBLs and transplanted with full-thickness allogeneic human skin. Human cell engraftment was enhanced by injection of anti-mouse CD122 antibody. The respective contributions of human CD4+ and CD8+ cells in allograft rejection were determined using depleting antibodies. Human skin grafts on unmanipulated NOD-scid B2mnull mice uniformly survived but on chimeric hu-PBL-NOD-scid B2mnull mice exhibited severe immune-mediated injury that often progressed to complete rejection. The alloaggressive hu-PBLs did not require prior in vitro sensitization to elicit targeted effector cell activity. Extensive mononuclear cell infiltration directed towards human-origin endothelium was associated with thrombosis and fibrin necrosis. No evidence of graft-versus-host disease was detected. Either CD4+ or CD8+ T cells may mediate injury and alloimmune rejection of human skin grafts on hu-PBL-NOD-scid B2mnull mice. It is proposed that Hu-PBL-NOD-scid B2mnull mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.     

Specific congenital heart defects in RSH/Smith-Lemli-Opitz syndrome: postulated involvement of the sonic hedgehog pathway in syndromes with postaxial polydactyly or heterotaxia

BACKGROUND

RSH/Smith‐Lemli‐Opitz syndrome is an autosomal recessive syndrome due to an inborn error of cholesterol metabolism and is characterized by developmental delay, facial anomalies, hypospadias, congenital heart defect (CHD), postaxial polydactyly, and 2–3 toe syndactyly. CHD is found in half of the propositi, and a specific association with atrioventricular canal defect (AVCD) and anomalous pulmonary venous return has been demonstrated.

METHODS

We report on an additional patient with RSH/SLOS presenting with complete AVCD and anomalous pulmonary venous return, and discuss the possible relationship of the Sonic Hedgehog (SHH) pathway as causative factor of these CHDs and those in heterotaxia patients with postaxial polydactyly syndromes.

RESULTS

Anatomic similarities between heterotaxia and CHDs of several syndromes with postaxial polydactyly have been noted previously, considering the frequent association of AVCD with common atrium in these conditions. It is known that both CHDs of heterotaxia and postaxial polydactyly can be related to abnormalities of the SHH pathway. Cholesterol has a critical role in the formation of normally active hedgehog proteins. It could be hypothesized that specific types of CHDs in RSH/SLOS can be caused by modifications of the SHH protein related to the defect of cholesterol biosynthesis.

CONCLUSIONS

The specific association of AVCD and anomalous pulmonary venous return in patients with RSH/SLOS and the finding of AVCD ± common atrium in several syndromes with polydactyly leads to the hypothesis that heterotaxia due to SHH anomalies could be involved in a large spectrum of conditions. Perturbations in different components of the SHH pathway could lead to several developmental errors presenting with partially overlapping clinical manifestations. Birth Defects Research (Part A) 67149–153, 2003. © 2003 Wiley‐Liss, Inc.

     

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.     

Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.