Nuclear translocation and signalling of L1-CAM in human carcinoma cells requires ADAM10 and presenilin/gamma-secretase activity

L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/gamma-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominantnegative PS1 or use of a specific gamma-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or gamma-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/gamma-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines. Key words: a disintegrin and metalloprotease 10 (ADAM10), L1 cell-adhesion molecule (L1-CAM), nuclear translocation, presenilin (PS)/gamma-secretase activity, raft, signalling

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P* becomes 4–5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll BA with its environment appears to be altered, possibly because of a change in its position

Nuclear translocation and signalling of L1-CAM in human carcinoma cells requires ADAM10 and presenilin/γ-secretase activity

L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/γ-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominant-negative PS1 or use of a specific γ-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or γ-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/γ-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines

ARLTS1 polymorphisms and basal cell carcinoma of the skin

Polymorphisms in the ARLTS1 gene, a member of the Ras super-family, have been associated with susceptibility in different cancer types. The involvement of the gene in apoptotic signalling motivated us to study the role of ARLTS1 polymorphic variations in basal cell carcinoma of the skin (BCC). In a case-control study, 529 cases diagnosed with BCC and 533 controls from Hungary, Romania and Slovakia were genotyped for the S99S (297G>A), P131L (392C>T), L132L (396G>C), C148R (442T>C) and W149X (446G>A) polymorphisms in the ARLTS1 gene. No significant association between any of the single nucleotide polymorphisms (SNP) and risk of BCC (S99S, odds ratio (OR) 0.96, 95% confidence interval (CI) 0.60-1.53; P131L, OR 1.31 95%CI 0.74-2.31; L132L, OR 0.50, 95%CI 0.02-7.07; C148R, OR 0.50, 95%CI 0.69-1.18; and W149X, OR 1.01, 95%CI 0.37-2.79) was detected. Furthermore, no significant difference in the distribution of haplotypes due to five polymorphisms in the ARLTS1 gene was found between the BCC cases and controls. Our data rule out an association between variants in ARLTS1 and risk of BCC in the investigated population

A Proton ENDOR Study of Azurin

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cβ atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein

Oxygen-sensitive 3He-MRI in bronchiolitis obliterans after lung transplantation

Oxygen-sensitive 3He-MRI was studied for the detection of differences in intrapulmonary oxygen partial pressure (pO2) between patients with normal lung transplants and those with bronchiolitis obliterans syndrome (BOS). Using software developed in-house, oxygen-sensitive 3He-MRI datasets from patients with normal lung grafts (n = 8) and with BOS (n = 6) were evaluated quantitatively. Datasets were acqiured on a 1.5-T system using a spoiled gradient echo pulse sequence. Underlying diseases were pulmonary emphysema (n = 10 datasets) and fibrosis (n = 4). BOS status was verified by pulmonary function tests. Additionally, 3He-MRI was assessed blindedly for ventilation defects. Median intrapulmonary pO2 in patients with normal lung grafts was 146 mbar compared with 108 mbar in patients with BOS. Homogeneity of pO2 distribution was greater in normal grafts (standard deviation pO2 34 versus 43 mbar). Median oxygen decrease rate during breath hold was higher in unaffected patients (−1.75 mbar/s versus −0.38 mbar/s). Normal grafts showed fewer ventilation defects (5% versus 28%, medians). Oxygen-sensitive 3He-MRI appears capable of demonstrating differences of intrapulmonary pO2 between normal lung grafts and grafts affected by BOS. Oxygen-sensitive 3He-MRI may add helpful regional information to other diagnostic techniques for the assessment and follow-up of lung transplant recipients

Defining DNA-based operational taxonomic units for microbial-eukaryote ecology

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 75 (2009): 5797-5808, doi:10.1128/AEM.00298-09.DNA sequence information has been increasingly used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, the autecology of ecologically relevant species, and the identification and enumeration of species of interest to human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is partly a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes, and partly the problematic nature and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximate species-level distinctions. The approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small subunit ribosomal RNA (18S) gene sequences and partial gene sequences obtained from GenBank for morphologically described protistan species. The program was tested using environmental 18S data sets from two oceanic ecosystems. A total of 388 operational taxonomic units were observed among 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific.Support for this manuscript was provided by National Science Foundation grants MCB-0732066, MCB-0703159 and OCE-0550829 and a grant from the Gordon and Betty Moore Foundation

Sensing the framework state and guest molecules in MIL-53(Al) via the electron paramagnetic resonance spectrum of V-IV dopant ions

X-ray diffraction (XRD) and electron paramagnetic resonance spectroscopy (EPR) were combined to study the structural transformations induced by temperature, pressure and air humidity of the "breathing'' metalorganic framework (MOF) MIL-53(Al), doped with paramagnetic V-IV ions, after activation. The correlation between in situ XRD and thermogravimetric analysis measurements showed that upon heating this MOF in air, starting from ambient temperature and pressure, the narrow pore framework first dehydrates and after that makes the transition to a large pore state (lp). The EPR spectra of V-IV=O molecular ions, replacing Al-OH in the structure, also allow to distinguish the as synthesized, hydrated (np-h) and dehydrated narrow pore (np-d), and lp states of MIL-53(Al). A careful analysis of EPR spectra recorded at microwave frequencies between 9.5 and 275 GHz demonstrates that all V-IV=O in the np-d and lp states are equivalent, whereas in the np-h state (at least two) slightly different V-IV=O sites exist. Moreover, the lp MIL-53(Al) framework is accessible to oxygen, leading to a notable broadening of the V-IV=O EPR spectrum at pressures of a few mbar, while such effect is absent for the np-h and np-d states for pressures up to 1 bar

Upper-Room Ultraviolet Light and Negative Air Ionization to Prevent Tuberculosis Transmission

Background Institutional tuberculosis (TB) transmission is an important public health problem highlighted by the HIV/AIDS pandemic and the emergence of multidrug- and extensively drug-resistant TB. Effective TB infection control measures are urgently needed. We evaluated the efficacy of upper-room ultraviolet (UV) lights and negative air ionization for preventing airborne TB transmission using a guinea pig air-sampling model to measure the TB infectiousness of ward air. Methods and Findings For 535 consecutive days, exhaust air from an HIV-TB ward in Lima, Perú, was passed through three guinea pig air-sampling enclosures each housing approximately 150 guinea pigs, using a 2-d cycle. On UV-off days, ward air passed in parallel through a control animal enclosure and a similar enclosure containing negative ionizers. On UV-on days, UV lights and mixing fans were turned on in the ward, and a third animal enclosure alone received ward air. TB infection in guinea pigs was defined by monthly tuberculin skin tests. All guinea pigs underwent autopsy to test for TB disease, defined by characteristic autopsy changes or by the culture of Mycobacterium tuberculosis from organs. 35% (106/304) of guinea pigs in the control group developed TB infection, and this was reduced to 14% (43/303) by ionizers, and to 9.5% (29/307) by UV lights (both p < 0.0001 compared with the control group). TB disease was confirmed in 8.6% (26/304) of control group animals, and this was reduced to 4.3% (13/303) by ionizers, and to 3.6% (11/307) by UV lights (both p < 0.03 compared with the control group). Time-to-event analysis demonstrated that TB infection was prevented by ionizers (log-rank 27; p < 0.0001) and by UV lights (log-rank 46; p < 0.0001). Time-to-event analysis also demonstrated that TB disease was prevented by ionizers (log-rank 3.7; p = 0.055) and by UV lights (log-rank 5.4; p = 0.02). An alternative analysis using an airborne infection model demonstrated that ionizers prevented 60% of TB infection and 51% of TB disease, and that UV lights prevented 70% of TB infection and 54% of TB disease. In all analysis strategies, UV lights tended to be more protective than ionizers. Conclusions Upper-room UV lights and negative air ionization each prevented most airborne TB transmission detectable by guinea pig air sampling. Provided there is adequate mixing of room air, upper-room UV light is an effective, low-cost intervention for use in TB infection control in high-risk clinical settings