Sulfamethoxazole biodegradation and impacts on soil microbial communities in a Bolivian arid high altitude catchment.

The processes controlling antibiotics fate in ecosystems are poorly understood, yet their presence can inhibit bacterial growth and induce the development of bacterial resistance. Sulfamethoxazole (SMX) is one of the most frequently detected sulfonamides in natural environments due to its low metabolism and molecular properties. This work presents pioneering results on SMX biodegradation and impact in high altitude soils (Bolivian Altiplano), allowing a better understanding of the persistence, spread and impact of this antibiotic at the global watershed scale. Our results showed significant dissipation of SMX in relation to its adsorption, hydrolysis and biotransformation. However, biodegradation appears to be lower in these mountain soils than in lowland soils as widely described in the literature. The half-life of SMX ranges from 12 to 346 days in non-sterile soils. In one soil, no biotic degradation was observed, indicating a likely high persistence. Biodegradation was related to OC content and to proximity to urban activities. Regarding the study of the impacts of SMX, the DGGE results were less sensitive than the sequencing. In general, SMX strongly changes the structure and composition of the studied soils at high altitudes, which is comparable to the observations of other authors in lowland soils. The phylum Actinobacter showed high sensitivity to SMX. In contrast, the abundance of ɣ-proteobacteria remained almost unchanged. Soil contamination with SMX did not lead to the development of the studied resistance genes (sul1 and sul2) in soils where they were absent at the beginning of the experiment. Thus, the presence of SMX resistance genes seems to be related to irrigation with wastewater carrying the studied resistance genes.

Antibiotic pollution in the Katari subcatchment of the Titicaca Lake: Major transformation products and occurrence of resistance genes.

An increasing number of studies pointed out the ubiquitous presence of medical residues in surface and ground water as well as in soil compartments. Not only antibiotics can be found in the environment but also their transformation products about which little information is generally available. The development of bacterial resistance to antibiotics is particularly worrying as it can lead to sanitary and health problems. Studies about the dissemination of antibiotics and associated resistances in the Bolivian Altiplano are scarce. We provide baseline information on the occurrence of Sulfamethoxazole (SMX) and Trimethoprim (TMP) antibiotics as well as on the most common human SMX transformation products (TP) and on the occurrence of sulfonamide resistance genes. The studied water and soil compartments presented high levels of antibiotic pollution. This situation was shown to be mainly linked with uncontrolled discharges of treated and untreated wastewaters, resulting on the presence of antibiotics in the Titicaca Lake. SMX TPs were detected in surface waters and on soil sampled next to the wastewater treatment plant (WWTP). SMX resistance genes sulI and sulII were widely detected in the basin hydrological network, even in areas unpolluted with antibiotics. Mechanisms of co-selection of antibiotic- and metal- resistance may be involved in the prevalence of ARG's in pristine areas with no anthropogenic activity and free of antibiotic pollution.

Hepatocyte growth factor protects hepatoblastoma cells from chemotherapy-induced apoptosis by AKT activation.

Hepatocyte growth factor/scatter factor (HGF) is a ubiquitously expressed molecule that elicits pleiotropic functions on epithelial cells, including mitogenic, motogenic, differentiating, angiogenic and morphogenic effects. In hepatoblastoma (HB), post-operative residual tumor growth and tumor recurrences are often associated with markedly elevated serum levels of HGF, suggesting a link between this molecule and tumor malignancy. Here, we demonstrate that HGF has no impact on overall cell viability and proliferation of HB cells, although signal transduction occurs downstream of HGF, such as c-Met phosphorylation, activation of phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK-1/2 signaling. Instead of being mitogenic, HGF confers anti-apoptotic properties upon serum starvation and moreover protects HB cells against strong apoptotic inducers such as cisplatin and camptothecin, thereby contributing to chemotherapeutic resistance. This effect is mainly dependent on the PI3K/AKT signaling pathway, since inhibition by wortmannin resulted in abrogation of HGF-mediated survival, whereas inhibition of the MAPK pathway had no effect. Together, these findings highlight the importance of HGF in tumor cell survival and suggest that HGF and its cognate receptor c-Met should be considered as a candidate for combined therapeutic strategies of advanced pediatric liver tumors.

Regulation of Pax3 transcriptional activity by SUMO-1-modified PML.

Pax3 is an evolutionarily conserved transcription factor that plays a major role in a variety of developmental processes. Mutations in Pax3 lead to severe malformations as seen in human Waardenburg syndrome and in the Splotch mutant mice. The transcriptional activity of Pax3 was recently shown to be repressed by Daxx whereas the oncogenic fusion protein Pax3-FKHR is unresponsive to this repressive action. Here we demonstrate that Daxx-mediated repression of Pax3 can be inhibited by the nuclear body (NB)-associated protein PML. Interestingly, this suppression of Daxx properties correlates with its recruitment to the NBs. Factors such as arsenicals and interferons that enhance NB formation, trigger both the targeting of Daxx to these nuclear structures and the relief of the repressive activity of Daxx. Conversely, lack of structurally intact NBs profoundly impairs Pax3 transcriptional activity, likely by increasing the pool of available nucleoplasmic Daxx. Moreover, a PML mutant that can not be modified by the ubiquitin-related SUMO-1 modifier is no more able to interact with Daxx. Consistently, such a mutant fails both to inhibit the Daxx repressing effect on Pax3 and to induce its accumulation into the NBs. Taken together, these results argue that SUMO-1 modified PML can derepress Pax3 transcriptional activity through sequestration of the Daxx repressor into the NBs and suggest a role for these nuclear structures in the transcriptional control by Pax proteins. Oncogene (2001) 20, 1 - 9.

c-Jun and p53 activity is modulated by SUMO-1 modification.

The ubiquitin-related SUMO-1 molecule has been shown recently to modify covalently a number of cellular proteins including IkappaBalpha. SUMO-1 modification was found to antagonize IkappaBalpha ubiquitination and protect it from degradation. Here we identify the transcription factors c-Jun and p53, two well known targets of ubiquitin, as new substrates for SUMO-1 both in vitro and in vivo. In contrast to ubiquitin, SUMO-1 preferentially targets a single lysine residue in c-Jun (Lys-229), and the abrogation of SUMO-1 modification does not compromise its ubiquitination. Activation of Jun NH(2)-terminal kinases, which induces a reduction in c-Jun ubiquitination, similarly decreases SUMO-1 modification. Accordingly, loss of the two major Jun NH(2)-terminal kinase phosphorylation sites in c-Jun, Ser-63 and Ser-73, greatly enhances conjugation by SUMO-1. A SUMO-1- deficient c-JunK229R mutant shows an increased transactivation potential on an AP-1-containing promoter compared with wild-type c-Jun, suggesting that SUMO-1 negatively regulates c-Jun activity. As with c-Jun, SUMO-1 modification of p53 is abrogated by phosphorylation but remains unaltered upon chemical damage to DNA or Mdm2-mediated ubiquitination. The SUMO-1 attachment site in p53 (Lys-386) resides within a region known to regulate the DNA binding activity of the protein. A p53 mutant, defective for SUMO-1 conjugation, shows unaltered ubiquitination but has a slightly impaired apoptotic activity, indicating that modification by SUMO-1 might be important for the full biological activity of p53. Taken together, these data provide a first link between the SUMO-1 conjugation pathway and the regulation of transcription factors.

Covalent modification of the transcriptional repressor tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies.

The ubiquitin-related SUMO-1 modifier can be covalently attached to a variety of proteins. To date, four substrates have been characterized in mammalian cells: RanGAP1, IkappaBalpha, and the two nuclear body-associated PML and Sp100 proteins. SUMO-1 modification has been shown to be involved in protein localization and/or stabilization and to require the activity of specialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologues have been identified in various species and belong to the so-called Smt3 family of proteins. Here we have characterized the Drosophila homologues of mammalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9, respectively). We show that dUbc9 is the conjugating enzyme for dSmt3 and that dSmt3 can covalently modify a number of proteins in Drosophila cells in addition to the human PML substrate. The dSmt3 transcript and protein are maternally deposited in embryos, where the protein accumulates predominantly in nuclei. Similar to its human counterpart, dSmt3 protein is observed in a punctate nuclear pattern. We demonstrate that Tramtrack 69 (Ttk69), a repressor of neuronal differentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Finally, we show that both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocalize on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 species can bind at sites of Ttk69 action in vivo. Altogether, these data indicate a high conservation of the Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of cell differentiation in Drosophila flies.