Evidence for a Xer/dif system for chromosome resolution in archaea

Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK. This chromosome resolution system has been predicted in most Bacteria and further characterized for some species. Archaea have circular chromosomes and an active homologous recombination system and should therefore resolve chromosome dimers. Most archaea harbour a single homologue of bacterial XerC/D proteins (XerA), but not of FtsK. Therefore, the role of XerA in chromosome resolution was unclear. Here, we have identified dif-like sites in archaeal genomes by using a combination of modeling and comparative genomics approaches. These sites are systematically located in replication termination regions. We validated our in silico prediction by showing that the XerA protein of Pyrococcus abyssi specifically recombines plasmids containing the predicted dif site in vitro. In contrast to the bacterial system, XerA can recombine dif sites in the absence of protein partners. Whereas Archaea and Bacteria use a completely different set of proteins for chromosome replication, our data strongly suggest that XerA is most likely used for chromosome resolution in Archaea.     

Characterization of missense mutations and large deletions in the ALPL gene by sequencing and quantitative multiplex PCR of short fragments

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.     

Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.     

Mutational analysis of the archaeal tyrosine recombinase SSV1 integrase suggests a mechanism of DNA cleavage in trans

The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases. This raised the possibility of a different mechanism for archaeal tyrosine recombinase. The residues of Int(SSV) tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied. The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases. In addition, we show that Int(SSV) exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils. Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity. This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer. The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase Int(SSV).     

A supramodal number representation in human intraparietal cortex

The triple-code theory of numerical processing postulates an abstract-semantic "number sense." Neuropsychology points to intraparietal cortex as a potential substrate, but previous functional neuroimaging studies did not dissociate the representation of numerical magnitude from task-driven effects on intraparietal activation. In an event-related fMRI study, we presented numbers, letters, and colors in the visual and auditory modality, asking subjects to respond to target items within each category. In the absence of explicit magnitude processing, numbers compared with letters and colors across modalities activated a bilateral region in the horizontal intraparietal sulcus. This stimulus-driven number-specific intraparietal response supports the idea of a supramodal number representation that is automatically accessed by presentation of numbers and may code magnitude information.     

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.     

Helicobacter pylori‐Associated Chronic Gastritis and Unexplained Iron Deficiency Anemia: a Reliable Association?

Background and aim. About 35% of iron deficiency anemia cases remain unexplained after a gastrointestinal evaluation. An association between Helicobacter pylori and iron malabsorption has been suggested. The aim of this study was to determine whether H. pylori‐associated chronic gastritis is linked to unexplained iron deficiency anemia in adults. Methods. From 1996 to 2001, we identified 105 patients with unexplained iron deficiency anemia after upper endoscopy, colonoscopy, small bowel radiographic examination and duodenal biopsies. Two biopsies were obtained from the gastric antrum and two from the corpus of each patient. Gastritis status was described according to the Sydney System and H. pylori infection was assessed by an immunohistochemical test on biopsy specimens. This group was compared to a control group matched for sex and age. Results. There were 76 women and 29 men (mean age 57.4 ± 21.4 years) examined in the study. A H. pylori‐associated chronic gastritis was identified in 63 cases (60%) vs. 45 cases (43%) cases in the control group (p < .01). Atrophic gastritis was significantly associated with iron deficiency anemia compared with the control group [16 (15%) vs. 6 (6%); p < .03]. In the unexplained iron deficiency anemia group, (1) patients with chronic gastritis were significantly younger (52 ± 22 vs. 64 ± 20 years; p < .005), and (2) chronic gastritis was not linked to sex [sex ratio (male/female): 0.5 vs. 0.34, p = .34]. The prevalence of H. pylori infection was similar between premenopausal and postmenopausal women [28 (27%) vs. 26 (25%); p = .7] with iron deficiency anemia. Conclusion. H. pylori infection and chronic gastritis, especially atrophic gastritis, are significantly associated with unexplained iron deficiency anemia. Relationships between H. pylori‐associated chronic gastritis and unexplained iron deficiency anemia should be considered.     

Aquatic insects as a vector for Mycobacterium ulcerans

Mycobacterium ulcerans is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking M. ulcerans to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of M. ulcerans, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that M. ulcerans may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition, M. ulcerans appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated M. ulcerans from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of M. ulcerans.