Eric Quiniou

Competition with Xenon Elicits Ligand Migration and Escape Pathways in Myoglobin

Evidence for ligand migration toward the xenon-binding cavities in myoglobin comes from a number of laser photolysis studies of MbO2 including mutants and from cryo- and time-resolved crystallography of MbCO. To explore ligand migration in greater detail, we investigated the rebinding kinetics of both MbO2 and MbCO under a xenon partial pressure ranging from 1 to 16 atm over the temperature range (293-77 K). Below 180 K xenon affects to a significant, but minor, extent the thermodynamic parameters for rebinding from the primary docking site in each Mb taxonomic substate. Above 200 K the ligand migrates to the proximal Xe1 site but when the latter is occupied by xenon a new kinetic process appears. It is attributed to rebinding from transient docking sites located on the path between the primary and the secondary docking site of both ligands. Ligand escape exhibits a more complicated pattern than expected. At room temperature O2 and CO escape appears to take place exclusively from the primary site. In contrast, at T approximately 250 K, roughly 50% of the CO molecules that have escaped from the protein originate from the Xe1 secondary site.

Radiation therapy and late mortality from second sarcoma, carcinoma, and hematological malignancies after a solid cancer in childhood.

PURPOSE To compare patterns of long-term deaths due to secondary carcinomas, sarcomas, and hematological malignancies occurring after childhood cancer in a cohort of patients followed over a median of 28 years. METHODS AND MATERIALS The study included 4,230 patients treated at eight institutions, who were at least 5-year survivors of a first cancer, representing 105,670 person-years of observation. Complete clinical, chemotherapeutic, and radiotherapeutic data were recorded, and the integral radiation dose was estimated for 2,701 of the 2,948 patients who had received radiotherapy. The integral dose was estimated for the volume inside the beam edges. The causes of death obtained from death certificates were validated. RESULTS In total, 134 events were due to second malignant neoplasm(s) (SMN). We found that the standardized mortality ratio decreased with increasing follow-up for second carcinomas and sarcomas, whereas the absolute excess risk (AER) increased for a second carcinoma but decreased for second sarcomas. There was no clear variation in SMN and AER for hematological malignancies. We found a significant dose-response relationship between the radiation dose received and the mortality rate due to a second sarcoma and carcinoma. The risk of death due to carcinoma and sarcoma as SMN was 5.2-fold and 12.5-fold higher, respectively, in patients who had received a radiation dose exceeding 150 joules. CONCLUSIONS Among patients who had received radiotherapy, only those having received the highest integral radiation dose actually had a higher risk of dying of a second carcinoma or sarcoma.

Long-term Mortality from Second Malignant Neoplasms in 5-Year Survivors of Solid Childhood Tumors: Temporal Pattern of Risk according to Type of Treatment

Background: The temporal pattern in mortality from late second malignant neoplasms in solid childhood cancer survivors, according to the type of treatment, has not been investigated in detail. Methods: We studied 4,230 5-year survivors of solid childhood cancer diagnosed between 1942 and 1986 in France and the United Kingdom. Complete clinical, chemotherapy, and radiotherapy data were recorded and the integral radiation dose was estimated for 2,701 of the 2,948 patients who had received radiotherapy. Results: After a median follow-up of 28 years, 134 fatal events were due to second malignancies, compared with the 13.3 expected from the general France-UK population rates. The standardized mortality ratio was of a similar magnitude after radiotherapy alone and chemotherapy alone and higher after both treatments. The standardized mortality ratio decreased with follow-up, whereas the absolute excess risk increased significantly over a period of at least 25 years after the first cancer. This temporal pattern was similar after chemotherapy alone, radiotherapy alone, or both treatments. We observed a similar long-term temporal pattern among survivors who had died of a second malignant neoplasm of the gastrointestinal tract and breast. Survivors who had received a higher integral radiation dose during radiotherapy were at a particularly high risk, as well as those who had received alkylating agents and epipodophyllotoxins. Conclusions: Five-year survivors of childhood cancer run a high long-term mortality risk for all types of second malignant neoplasms whatever the treatment received and require careful long-term screening well beyond 25 years after the diagnosis. Cancer Epidemiol Biomarkers Prev; 19(3); 707–15

Phenylisoserine: A Versatile Amino Acid for the Construction of Novel β-Peptide Structures

The N-Boc O-tert-butyldimethysilyl-substituted hexa-beta-peptide methyl ester 18 was constructed from the O-TBS ether of (-)-(2R, 3S)-phenylisoserine. By NMR, it was determined that this homo beta-peptide adopts a highly stable beta-strand-type secondary structure in chloroform solution, which is stabilized by both hydrophobic interactions involving the OTBS methyl groups of residues i and i + 2, and inter-(five-membered)/intra (six-membered)-residue H-bonding interactions. These interactions are systematically repeated along the peptide chain and, thereby, operate in concert to stabilize the observed conformation of 18.

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

SummaryDynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from 13C relaxation rates R1, R2 and steady-state 13C-{1H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping Cα resonances and for 11 threonine Cβ single resonances. Except for three Cα relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C−H fragments exhibit fast (τe < 40 ps) restricted libration motions (S2=0.73 to 0.95). Global examination of the microdynamical parameters S2 and τe along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The β-ribbon comprising residues 37 to 47 is spatially restricted, with relatively large τe values in its hairpin region. The other β-ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala1 and Ala2, and the C-terminal residue Asn113, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain Cβ must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr65, Thr68, Thr81) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S2, τe and R2ex) or (Sinfslowsup2, Sinffastsup2and τslow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time τe for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1−S2)× τe is nearly independent of τe for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.

Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: Evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations

The three‐dimensional structure of apo‐neocarzinostatin (apo‐NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven‐stranded antiparallel β‐sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo‐NCS by 13C‐NMR relaxation measurements and molecular dynamics simulation. Model‐free parameters determined from the experimental data are compared with a 1.5‐nsec molecular simulation of apo‐NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this β‐protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

Role of radiation dose in the risk of secondary leukemia after a solid tumor in childhood treated between 1980 and 1999.

PURPOSE The purpose of this study was to estimate the risk of secondary leukemia as a function of radiation dose, taking into account heterogeneous radiation dose distribution. METHODS AND MATERIALS We analyzed a case-control study that investigated the risk of secondary leukemia and myelodysplasia after a solid tumor in childhood; it included 61 patients with leukemia matched with 196 controls. Complete clinical, chemotherapy, and radiotherapy histories were recorded for each patient in the study. Average radiation dose to each of seven bone marrow components for each patient was incorporated into the models, and corresponding risks were summed up. Conditional maximum likelihood methods were used to estimate risk parameters. RESULTS Whatever the model, we failed to evidence a role for the radiation dose to active bone marrow in the risk of later leukemia, myelodysplasia, or myeloproliferative syndrome, when adjusting for epipodophyllotoxin and anthracycline doses. This result was confirmed when fitting models that included total dose of radiation delivered during radiotherapy, when fitting models taking into account dose per fraction, and when restricting the analysis to acute myeloid leukemia. CONCLUSIONS In contrast to results found in similar studies that included children treated before the use of epipodophyllotoxins, this study failed to show a role for radiotherapy in the risk of secondary leukemia after childhood cancer in children treated between 1980 and 1999. This discrepancy was probably due to a competitive mechanism between these two carcinogens.

What determines the degree of compactness of a calcium-binding protein?

The EF‐hand calcium‐binding proteins may exist either in an extended or a compact conformation. This conformation is sometimes correlated with the function of the calcium‐binding protein. For those proteins whose structure and function are known, calcium sensors are usually extended and calcium buffers compact; hence, there is interest in predicting the form of the protein starting from its sequence. In the present study, we used two different procedures: one that already exists in the literature, the sosuidumbbell algorithm, mainly based on the charges of the two EF‐hand domains, and the other comprising a novel procedure that is based on linker average hydrophilicity. The linker consists of the residues that connect the domains. The two procedures were tested on 17 known‐structure calcium‐binding proteins and then applied to 59 unknown‐structure centrins. The sosuidumbbell algorithm yielded the correct conformations for only 15 of the known‐structure proteins and predicted that all centrins should be in a closed form. The linker average hydrophilicity procedure discriminated well between all the extended and non‐extended forms of the known‐structure calcium‐binding proteins, and its prediction concerning centrins reflected well their phylogenetic classification. The linker average hydrophilicity criterion is a simple and powerful means to discriminate between extended and non‐extended forms of calcium‐binding proteins. What is remarkable is that only a few residues that constitute the linker (between 2 and 20 in our tested sample of proteins) are responsible for the form of the calcium‐binding protein, showing that this form is mainly governed by short‐range interactions.

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

SummaryNearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural 13C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH2 side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, 13C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.