Modelling of marine debris pathways into UK waters: Example of non-native crustaceans transported across the Atlantic Ocean on floating marine debris.

The long-distance transfer of non-native, potentially invasive species via floating marine debris is an increasing threat to biodiversity and conservation efforts. To address the lack of understanding around mechanisms and pathways of species transfer via marine debris, a novel modelling approach was applied to recreate the likely trajectory and source of a large piece of debris fouled by non-native species collected from UK marine waters. This approach applied the Oil Spill Contingency and Response (OSCAR) simulation tool, an adapted oil spill modelling programme, which was informed by a combination of biological trait information for the foulant species, marine debris characteristics and hydrodynamic data. The modelling output suggested an origin in the Western Atlantic, a scenario concurrent with the known distribution of the foulant species. This modelling approach represents a valuable tool with which to determine the origin and trajectory of invasive species transferred via marine debris.

Clinical Phenotype in an Early-Onset French Pediatric Population: Charcot-Marie-Tooth's Disease Type 2A.

Charcot-Marie-Tooth's disease type 2A (MCT2A), induced by mutation of the mitofusin 2 (MFN2) gene represents the main cause of MCT2. The aim of this study is to provide details of the clinical and electromyographic phenotype of MCT2A in a pediatric population. We conducted a French multicenter retrospective study, including all children with a genetic diagnosis of MCT2A. Thirteen MCT2A children were included with a beginning of symptoms before the age of 10 years ("early-onset group"). We report two new mutations: c.1070 A → T (p.Lys357.Met) and c.280 C → G (p.Arg94Gly). The evolution of the disease is marked by a fast worsening for three patients with loss of motor autonomy, while the evolution is relatively stable for eight patients. The group of early-onset MCT2A seems more heterogeneous than previously described, with a nonconstant severe phenotype.

LIM-class homeobox gene Lim1, a novel oncogene in human renal cell carcinoma.

Human clear cell renal cell carcinoma (CCC) remains resistant to therapies. The transcription factor LIM-class homeobox gene Lim1 is required for normal organogenesis, including nephrogenesis, by regulating cell movements, differentiation and growth. Its expression is controlled partly by the sonic hedgehog-Gli signaling pathway, which we have recently shown to be reactivated in human CCC. So far, no study has assessed whether Lim1 may be associated with tumorigenesis. Using a panel of human CCC cell lines expressing or not the von Hippel-Lindau tumor suppressor gene and 44 tumor/normal tissues pairs, we found that Lim1 is constitutively and exclusively reexpressed in tumors. Through Lim1 silencing or overexpressing, we show that Lim1 is a growth and survival factor in human CCC, at least through the activation of oncogenic pathways including the phosphoinositide kinase-3/Akt and nuclear factor-kappaB pathways. More importantly, in nude mice bearing human CCC tumors, Lim1 silencing abolished tumor growth through the same mechanism as in vitro. In Lim1-depleted cells and tumors, cell movements were substantially impaired because of the inhibition of expression of various proteins involved in metastatic spread, such as paxillin or tenascin-C. These findings establish that the developmental marker Lim1 acts as an oncogene in cancer cells and targeting Lim1 may constitute an innovative therapeutic intervention in human CCC.

Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker.

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation.

Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-kappaB, involving phosphorylation of its inhibitor IkappaB-alpha on tyrosine 42. This modification does not lead to degradation of IkappaB by the proteasome/ubiquitin pathway, as is seen on stimulation of cells with proinflammatory cytokines. It is currently unknown how tyrosine-phosphorylated IkappaB is removed from NF-kappaB. Here we show that p85alpha, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IkappaB-alpha in vitro and in vivo after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IkappaB is sequestered from NF-kappaB. Another mechanism by which PI3-kinase contributed to NF-kappaB activation in response to pervanadate appeared to involve its catalytic p110 subunit. This was evident from the inhibition of pervanadate-induced NF-kappaB activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound had virtually no effect on tumor necrosis factor- and interleukin-1-induced NF-kappaB activities. Wortmannin did not inhibit tyrosine phosphorylation of IkappaB-alpha or alter the stability of the PI3-kinase complex but inhibited Akt kinase activation in response to pervanadate. Our data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-kappaB activation by the tyrosine phosphorylation-dependent pathway.

Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction.

The human T-cell leukemia virus type I (HTLV-I) has been etiologically associated with the development of the adult T-cell leukemia (ATL) as well as degenerative neurologic syndrome termed tropical spastic paraparesis (TSP). HTLV-I encodes a potent transactivator protein termed Tax that appears to play an important role in the process of T-cell immortalization. Even though the mechanisms by which Tax induces transformation are still unknown, it seems likely that the ability of Tax to alter the expression of many cellular genes plays an important part in this process. Tax does not bind directly to DNA but rather deregulates the activity of cellular transcription factors. One family of host transcription factors whose activity is altered by Tax includes NF-kappa B/Rel. These transcription factors are post-transcriptionally regulated by their assembly with a second family of inhibitory proteins termed I kappa B that serve to sequester the NF-kappa B/Rel complexes in the cytoplasm. Upon cellular activation, I kappa B alpha is phosphorylated, polyubiquitinated, and degraded in the proteasome. This proteolytic event liberates NF-kappa B, permitting its rapid translocation into the nucleus where it binds to its cognate enhancer elements. Similarly, the p105 precursor of the NF-kappa B p50 subunit is also post-translationally processed in the proteasome. The mechanisms by which Tax activates NF-kappa B remain unclear, and findings presented in the literature are often controversial. We identified a physical interaction between Tax and the HsN3 subunit of the human proteasome. This raises the intriguing possibility that physical association of the HsN3 proteasome subunit with HTLV-I Tax coupled with the independent interaction of Tax with either p100 or p65-I kappa B alpha targets these cytoplasmic NF-kappa B/Rel complexes to the proteasome for processing.

Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene.

The tax gene product of human T-cell leukemia virus type I (HTLV-I) is a potent transcriptional activator that both stimulates viral gene expression and activates an array of cellular genes involved in T-cell growth. Tax acts indirectly by inducing or modifying the action of various host transcription factors, including members of the NF-kappa B/Rel family of enhancer-binding proteins. In resting T cells, many of these NF-kappa B/Rel factors are sequestered in the cytoplasm by various ankyrin-rich inhibitory proteins, including I kappa B alpha. HTLV-I Tax expression leads to the constitutive nuclear expression of biologically active NF-kappa B and c-Rel complexes; however, the biochemical mechanism(s) underlying this response remains poorly understood. In this study, we demonstrate that Tax-stimulated nuclear expression of NF-kappa B in both HTLV-I-infected and Tax-transfected human T cells is associated with the phosphorylation and rapid proteolytic degradation of I kappa B alpha. In contrast to prior in vitro studies, at least a fraction of the phosphorylated form of I kappa B alpha remains physically associated with the NF-kappa B complex in vivo but is subject to rapid degradation, thereby promoting the nuclear translocation of the active NF-kappa B complex. We further demonstrate that Tax induction of nuclear c-Rel expression is activated by the RelA (p65) subunit of NF-kappa B, which activates transcription of the c-rel gene through an intrinsic kappa B enhancer element. In normal cells, the subsequent accumulation of nuclear c-Rel acts to inhibit its own continued production, indicating the presence of an autoregulatory loop. However, the pathologic action HTLV-I Tax leads to the deregulated and sustained nuclear expression of both NF-kappa B and c-Rel, a response that may contribute to HTLV-I-induced T-cell transformation.

Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs.

RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B. The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for I kappa B alpha binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B.

Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product: implications for viral latency.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, Tax is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors HEB-1 and p67SRF. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I Tax can physically associate with p100, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein. In contrast, a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100. Together, these results suggest that the cytoplasmic interplay of Tax and p100 may play an important role in the initiation and maintenance of HTLV-1 latency observed in adult T-cell leukemia.

Functional and biochemical interaction of the HTLV-I Tax1 transactivator with TBP.

The human T-cell leukemia virus type I (HTLV-I) codes for the potent transcriptional activator, Tax1, which induces the enhancer activity of various enhancer elements. In the case of the 21 bp enhancer of the HTLV-I provirus, this induction is correlated with the association of Tax1 with this DNA element via a specific cellular factor. That the indirect association of Tax1 with DNA can lead to transcriptional activation has also been supported by the study of chimeric GAL4-Tax1 proteins. The GAL4-Tax1 stimulatory effect exhibits a strong self-squelching. In order to determine whether Tax1 interacts directly with the general transcription factors or via intermediary molecules, we have analyzed how overexpression of the TATA binding protein (TBP) and TFIIB protein affects the squelching curve of GAL4-Tax1. The data presented here show that overexpression of TBP strongly increases the stimulatory effect of GAL4-Tax1, causes a displacement of the maximum of the squelching curve and partially alleviates the squelching. Under similar conditions TFIIB exhibited little effect. From these results we conclude that Tax1 can increase the recruitment of TBP by directly interacting with this protein. Biochemical experiments with purified proteins produced in bacteria confirmed that Tax1 can interact with TBP but not with TFIIB. Tax1 interacts with the conserved C-terminal part of TBP. Analysis of the ability of different mutants of Tax1 fused to the GAL4 DNA binding domain to activate transcription and to associate with TBP, showed that these activities are correlated. However, since one transcriptionally inactive mutant was able to interact efficiently with TBP in vitro, it would appear that an event other than the Tax1-TBP contact also intervenes in the activation of transcription by Tax1.

The transcriptionally active factors mediating the effect of the HTLV-I Tax transactivator on the IL-2R alpha kappa B enhancer include the product of the c-rel proto-oncogene.

The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor (IL-2R alpha) via a kappa B site that can bind several protein species of the rel family. Tax1 strongly activates the enhancer activity of this motif, in both epithelial HeLa and lymphoid Jurkat cells. This activation was not observed in undifferentiated embryocarcinoma F9 cells. Overexpression of the p50, p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65. Moreover, whereas both Tax and phorbol 12-myristate 13-acetate (PMA) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site, PMA is functionally inactive. Using the DNA affinity precipitation assay, we observed that Tax1 is able to efficiently induce the binding of Rel, whereas PMA is not. This established a clear difference between both stimuli, indicating that Rel is the functionally active factor. We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel can be a potent transcriptional activator on specific kappa B sites.

Binding of the HTLV-I Tax1 transactivator to the inducible 21 bp enhancer is mediated by the cellular factor HEB1.

Transcription driven by the HTLV-I promoter is strongly activated by the viral transactivator protein Tax1. This effect is mediated via a 21 bp sequence which is imperfectly repeated three times in the viral promoter. We showed previously that a single 21 bp copy exhibits a strong Tax1-inducible enhancer activity and is able to bind different cellular proteins, namely ATF, HEB1 and HEB2. We have further investigated the molecular mechanism involved in the Tax1 induction of the 21 bp motif's enhancer activity by analysing Tax1 interaction with this DNA sequence. For this purpose a HeLa cell line constitutively expressing a functional Tax1 protein was established and nuclear extracts of these cells were used to perform a DNA affinity precipitation assay. This experimental approach allowed us to show that Tax1 specifically binds to the 21 bp motif. The same sequence elements of the 21 bp motif are required both for Tax1 binding and for Tax1-induced enhancer activity. Chromatographic fractionation of the HeLa tax nuclear extract showed that the binding is indirect and is mediated by the cellular factor HEB 1.

Tax1 induction of the HTLV-I 21 bp enhancer requires cooperation between two cellular DNA-binding proteins.

Activation of the HTLV-I promoter by the viral Tax1 transactivator is mediated by a 21 bp sequence motif imperfectly repeated three times and composed of three exactly conserved domains (A, B and C from 5' to 3'). We show here that the Tax1 response requires the integrity of the B domain and of at least one of the flanking A or C domains. We have identified three cellular proteins which bind specifically to the 21 bp motif. One of these is the already well-characterized transcription factor ATF. The other two, namely HEB1 and HEB2, are specific for the 21 bp motif. HEB1 can bind to either domain A or C, but binding of ATF and HEB2 is determined by domain B. However, neither domain B alone, nor ATF/CREB binding sites respond significantly to Tax1. We therefore propose that Tax1 induction of the 21 bp enhancer element requires interaction with the two different cellular proteins identified in this study: HEB1 and HEB2, rather than binding of the ATF factor.

[Evaluation of decontamination procedures used at digestive endoscopy units in Gironde]. / Evaluation des procédures de décontamination utilisées dans les centres d'endoscopie digestive de Gironde.

Decontamination procedures used for endoscopes were noted in 23 digestive endoscopy units, public and private, in the department of Gironde and compared to recommended procedures. Serial, bacteriological samples were obtained from one esogastroscope and one colonoscope in each unit, after upper endoscopy and colonoscopy diagnostic procedures at the end of the endoscopy session. Six units of 23 used complete decontamination procedures. In the 17 other units, principal errors of decontamination procedures were: inadequate cleaning of internal channel of scopes (12 units) and lack of utilization of glutaraldehyde between each endoscopy (8 units). Bacteriological samples were negative in 11/12 endoscopes after a complete decontamination procedure and in 8/39 after an inadequate procedure (p less than 0.01). Complete procedures are efficacious but not used often enough. Information and changes in endoscopic practices are necessary in digestive endoscopy units.