Crystal structure of the active form of native human thymidylate synthase in the absence of bound substrates.

Human thymidylate synthase (hTS) provides the sole de novo intracellular source of thymidine 5'-monophosphate (dTMP). hTS is required for DNA replication prior to cell division, making it an attractive target for anticancer chemotherapy and drug discovery. hTS binds 2'-deoxyuridine 5'-monophosphate (dUMP) and the folate co-substrate N5,N10-methylenetetrahydrofolate (meTHF) in a pocket near the catalytic residue Cys195. The catalytic loop, which is composed of amino-acid residues 181-197, can adopt two distinct conformations related by a 180° rotation. In the active conformation Cys195 is close to the active site, while in the inactive conformation it is rotated and Cys195 is too distant from the active site for catalysis. Several hTS structures, either native or engineered, have been solved in the active conformation in complex with ligands or inhibitors and at different salt concentrations. However, apo hTS structures have been solved in an inactive conformation in high-salt and low-salt conditions (PDB entries 1ypv, 4h1i, 4gyh, 3egy and 3ehi). Here, the structure of apo hTS crystallized in the active form with sulfate ions coordinated by the arginine residue that binds dUMP is reported.

Differences between nicotine-abstinent smokers and non-smokers in terms of visuospatial attention and inhibition before and after single-blind nicotine administration.

The cholinergic system is implicated in visuospatial attention and inhibition, however the exact role is still unclear. Two key mechanisms in visuospatial attention are bias and disengagement. Bias refers to neuronal signals that enhance the sensitivity of the sensory cortex, disengagement is the decoupling of attention. Previous studies suggest that nicotine affects disengagement and (related) inhibition. However the exact relation is still unknown. Furthermore, nicotine-abstinence in 'healthy' smokers may resemble some anomalies of visuospatial attention and inhibition as seen in Attention Deficit/Hyperactivity Disorder (ADHD). Smokers and non-smokers (32 male students) performed in a visuospatial cueing (VSC) task, to assess bias and disengagement, and in a stop-signal task (SST) to assess inhibition. It was expected that nicotine abstinent smokers compared to non-smokers, would show poor disengagement (indicated by an enhanced validity effect) and poor inhibitory control (indicated by an enhanced stop-signal reaction time (SSRT)). It was expected that nicotine would positively affect disengagement and inhibition: hypothesis 1 stated that this effect would be larger in smokers as opposed to non-smokers, in terms of smoking-related deficient inhibitory control. Hypothesis 2 stated the exact opposite, in terms of drug-tolerance. Results indicated no baseline differences. Nicotine enhanced inhibition more in non-smokers relative to smokers. Integrating the results, nicotine-abstinent smokers do not seem to resemble ADHD patients, and do not seem to smoke in order to self-medicate a pre-existing deficit pertaining to mechanisms of visuospatial attention and inhibition. Nicotine may affect inhibition more in non-smokers relative to smokers, consistent with a drug-tolerance account.

The effect of enhancing cholinergic neurotransmission by nicotine on EEG indices of inhibition in the human brain.

The role of the cholinergic system in inhibition remains to be elucidated. Nicotine is a potent tool to augment this system, but most studies investigated its effects solely on behavior. Reference to brain activity is important to specifically identify inhibition-related mechanisms. In the current study the objective was to elucidate the role of the cholinergic system in inhibition. 16 healthy non-smokers performed in a stop task while EEG was recorded. A pre- versus post-treatment, within subjects, placebo controlled, single-blind design was used. It was hypothesized that nicotine would decrease stop-signal reaction time (SSRT) and increase the amplitude of inhibition-related event related potentials, the stop N2 and stop P3. Behavioral measures show nicotine shortened SSRT, but only when pretreatment values were not taken into account. On EEG measures, an enhanced stop P3 under nicotine was found, but only in a subsample sensitive to nicotine based on diastolic blood pressure. The results are indicative of enhanced inhibitory activity possibly reflecting enhanced activation in the superior frontal gyrus.

The effect of the augmentation of cholinergic neurotransmission by nicotine on EEG indices of visuospatial attention.

The cholinergic system has been implicated in visuospatial attention but the exact role remains unclear. In visuospatial attention, bias refers to neuronal signals that modulate the sensitivity of sensory cortex, while disengagement refers to the decoupling of attention making reorienting possible. In the current study we investigated the effect of facilitating cholinergic neurotransmission by nicotine (Nicorette Freshmint 2mg, polacrilex chewing gum) on behavioral and electrophysiological indices of bias and disengagement. Sixteen non-smoking participants performed in a Visual Spatial Cueing (VSC) task while EEG was recorded. A randomized, single-blind, crossover design was implemented. Based on the scarce literature, it was expected that nicotine would specifically augment disengagement related processing, especially manifest as an increase of the modulation of the Late Positive Deflection (LPD) by validity of cueing. No effect was expected on bias related components (cue-locked: EDAN, LDAP; target-locked: P1 and N1 modulations). Results show weak indications for a reduction of the reaction time validity effect by nicotine, but only for half of the sample in which the validity effect on the pretest was largest. Nicotine reduced the result of bias as indexed by a reduced P1 modulation by validity, especially in subjects with strong peripheral responses to nicotine. Nicotine did not affect ERP manifestations of the directing of bias (EDAN, LDAP) or disengagement (LPD).

[Reversible metalation of a bis-disulfide analogue of the Cys*-X-Cys* hepcidin binding site: structural characterisation of the related copper complex]. / Métallation réversible d'un analogue bis-disulfure du site de liaison Cys*-X-Cys* de l'hepcidine: caractérisation structurale du complexe de cuivre associé.

Hepcidin, a 25-amino-acid peptide secreted by the liver, distributed in the plasma and excreted in urine, is a key central regulator of body iron homeostasis. This hormone decreases export of cellular iron by binding to ferroportin, an iron exporter present at the basolateral surface of enterocytes and macrophages (the sites of dietary iron absorption and iron recycling, respectively), inducing its internalization and degradation. Hepcidin contains eight cysteine residues that form four disulfide bridges, which stabilize a hairpin-shaped structure with two beta sheets. We noticed in the sequence of hepcidin a Cys*-X-Cys* motif which can act as a metal binding site able to trap iron and/or copper. We have tested this hypothesis using a pseudopeptidic synthetic bis-disulfide analogue and we have shown that direct metalation of such ligand leads to the formation of a copper(III) complex with the typical N(2)S(2) donor set. This compound crystallizes in the orthorhombic system, space group Imma. The Cu(III) configuration is square planar, built up from two carboximado-N and two thiolato-S donors. This complex is converted back to the bis-disulfide, with release of the copper salt, upon oxidation with iodine.

Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair.

Acute promyelocytic leukaemia (APL) is a distinctive subtype of acute myeloid leukaemias. Even through this human disease can be treated by the intravenous administration of all-trans retinoic acid (ATRA), 25% of patients typically relapse after the first treatment. In this context, the intravenous administration of APL patients with an aqueous solution of arsenic trioxide has also been demonstrated to be successful despite the established mammalian toxicity of this arsenic compound. Accordingly, the administration of a therapeutic dose of arsenic trioxide has resulted in an improved patient survival in both relapsing as well newly diagnosed APL patients. We present here a mini-review of the medicinal use of arsenite, its mammalian metabolism (with an emphasis on biomethylation pathways), its elimination and pharmacokinetics and the novel application of hair analysis as a biomonitoring material. This mini-review also introduces our own results on the analysis of hair of patients receiving arsenic trioxide therapy. In this work, instead of quantifying arsenic content in bulk hair, we performed longitudinal analysis in order to use hair as a marker of arsenic exposure correlated to a time scale. Taking into account the hair growth rate, the longitudinal analysis of hair is demonstrated to provide a chronological record of the treatment of patients with arsenic trioxide. The small quantity of material to be analysed required the use of Synchrotron radiation based X-ray fluorescence (SXRF) spectroscopy. The hair arsenic content was well correlated with the clinical background of patients and reflected the intake of arsenic trioxide. In particular, the onset of arsenic trioxide therapy and interruptions during therapy were reflected by total arsenic content, which suggested rapid elimination. Another type of experiment, micro-XRF cartography on thin hair slices, allowed us to obtain distribution maps of arsenic, which demonstrated that arsenic is located at the periphery of hair. Micro-XANES spectra recorded at the periphery of hair, suggest that inorganic arsenic is incorporated in hair in its trivalent oxidation state, in agreement with previous results.

[Proposal for a discriminant level of BNP in very elderly persons with heart failure]. / Proposition d'un seuil de BNP discriminant dans la population très âgée présentant une insuffisance cardiaque.

A diagnosis of heart failure (HF) in elderly patient can be difficult. The purpose of this study is to evaluate the cut-off of brain natriuretic peptide (BNP) Triage Assay (Biosite) in the diagnosis of HF in this population : 250 very older patients with age

XAS applied to pharmaceuticals: drug administration and bioavailability.

We present selected XAS applications, focused towards practical hospital questions of drug administration and bioavailability, where the technique is driven up to its limits of sensitivity. i) XAS was used to study the interactions between the components of parenteral nutrition solutions, in particular zinc and aminoacids, possibly modifying their bioavailability. ii) We studied by EXAFS a series of binary and ternary copper-aminoacid complexes, in view of the development of an efficient oral drug against copper deficiencies in Menkes disease. iii) EXAFS and XANES analysis allowed us to characterise the solution form of a new arsenic containing drug against leukaemia. In parallel to the XAS measurements, we analysed trace elements levels along patients' hairs, using X-ray fluorescence excited by synchrotron radiation. The measurements along the hair allow for a monitoring of essential trace elements during therapy.

Stimulation of osteoclast differentiation in vitro by mouse oncostatin M, leukaemia inhibitory factor, cardiotrophin-1 and interleukin 6: synergy with dexamethasone.

The role of oncostatin M in bone metabolism is not clearly defined, and the actions of mouse oncostatin M (mOSM) on osteoclast development has not been previously determined. We therefore examined the ability of recombinant mOSM to stimulate osteoclast formation and activity using cocultures of murine calvaria and bone marrow cells, and compared the responses to other members of the interleukin 6 family of cytokines including mouse leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and IL-6. Mouse OSM, LIF and CT-1 strongly induced the formation of tartrate resistant acid phosphatase positive (TRAP(+)) multinucleated cells (MNC) in a dose-dependent fashion. OSM, LIF or CT-1 also elevated the number and size of resorptive pits when cocultures were added to smooth cortical bone slices, indicating enhancement of osteoclast activity. The activity of OSM was reduced by indomethacin (10(-8)-10(-6) M), whereas addition of dexamethasone (DEX) at 10(-7)-10(-5) M synergistically enhanced OSM-induced numbers of TRAP(+)MNC. DEX (10(-7) M) costimulation also synergistically enhanced TRAP(+)cell numbers of LIF, and CT-1 treated cocultures. IL-6 had no activity alone, but further enhanced TRAP(+)cell formation in mOSM or DEX (10(-7) M) treated cocultures. When added to mouse calvarial osteoblast cultures, mOSM induced secretion of IL-6 protein and elevation of mRNA whereas LIF or CT-1 did not. IL-6 mRNA levels and protein secretion were reduced in osteoblasts by costimulation with DEX. These results show that mouse OSM, LIF and CT-1 induce osteoclast differentiation and activation, that DEX synergizes with each in this activity, and that mouse OSM induces responses in osteoblasts that are not shown by LIF or CT-1. Collectively these data suggest an important role of these cytokines in osteoporosis caused by high levels of corticosteroid.

Submicrometre beams from a hard X-ray waveguide at a third-generation synchrotron radiation source.

The use of an X-ray waveguide for scattering experiments at an undulator of a third-generation synchrotron radiation source is discussed. The performance with a perfect crystal monochromator, multilayer monochromator and focusing mirror is explored. A maximum flux of 8 x 109 photons s(-1) at lambda = 0.083 nm was obtained for a 0.15 (V) x 600 (H) micron(2) beam at the exit of the waveguide with a multilayer monochromator. The combination of an Si (111) monochromator and ellipsoidal mirror resulted in a flux of approximately 10(9) photons s(-1) but with a horizontal compression of the beam to approximately 30 micron. The use of the waveguide in diffraction experiments is addressed.

[Physiopathology of bedsores]. / Physiopathologie des escarres.

Pressure is the primary pathogenic factor in the development of decubitus ulcers. Other major factors are shearing forces, friction and moisture. Significant intrinsic risk factors are immobility, age-related diseases, nutritional status, medications and smoking. The morbidity and mortality related to the complications of pressure sores are quite significant. Prevention is essential and is best achieved by identification of high risk patients. The therapeutic approach is based on the grade of pressure ulcer.

Adult onset Kawasaki disease diagnosed by the echocardiographic demonstration of coronary aneurysms.

A 17-year-old boy presented with fever, bilateral conjunctival infection, angina and extensive cervical adenopathy. Amoxycillin was started. Ten days later he was admitted to hospital because of persistent high fever, cervical adenopathy, erythema of the pharynx and tongue and lip fissuration. The most important interventions of his first hospitalization were endotracheal intubation because of increasing dyspnoea due to adult respiratory distress syndrome and haemodialysis for renal insufficiency. His admission to our hospital was marked by the echocardiographic discovery of giant coronary aneurysms in the first few centimeters of both right and left coronary arteries. Coronary angiography confirmed giant aneurysm formation of the right and left coronary arteries. Similarly, medium sized arteries (cerebral, hepatic, mesenteric, iliac) presented abnormalities and laboratory findings. This is the first description of adult-onset Kawasaki disease with giant coronary aneurysm formation and more generalized arterial involvement. The severity of the clinical symptoms and the severity of the coronary disease indicates that Kawasaki disease of the adult does not always have a benign course.

Resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and Chinese hamster ovary-multi-drug resistant. Cells in vitro.

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.

[Buerger's disease. Clinical and therapeutic review. Apropos of a case]. / La maladie de Buerger. Rappel clinique et thérapeutique. A propos d'un cas.

In reference to a typical case of Buerger's disease, the clinical, arteriographic, histological signs as well as diagnostic criteria are restated. The pathogenesis is still unknown; a genetic predisposition and an auto-immune process could be involved. The treatment, presently symptomatic, cannot arrest the evolution which is often mutilating, when the patient continues to smoke. An early diagnosis is possible in the presence of superficial phlebitis or Raynaud's phenomenon.

Keratoderma climactericum (Haxthausen's disease): clinical signs, laboratory findings and etretinate treatment in 10 patients.

10 cases of keratoderma climactericum are reported. This keratosis of the palms and soles appears late in women of menopausal age. The keratotic lesions first develop at the plantar pressure points, making walking troublesome. Involvement of the hands remains discrete. Examination for contact allergy, fungal tests, vitamin A serum levels, and sex hormones were negative or normal in all the 10 patients. Microscopy revealed a lichenified eczema with evidence of mechanical irritation. Etretinate (0.78 mg/kg/day) brought about partial or total remission of the hyperkeratosis. Pain on walking disappeared in all the patients.

[Interaction between asthma and maxillary sinusitis]. / Interaction entre asthme et sinusite maxillaire.

We have studied atopic status and respiratory function in 69 asthmatics in relation to possible radiological changes in the sinuses. These alterations were classified under four headings: normal, mucosal thickening (greater than 3 mm), polyp or opaque films. The bronchial obstruction, evaluated by FEV1/FVC ratio was compared in the 4 groups. The reversibility of airflow obstruction by beta-2-sympathomimetics (Fenoterol) was the same with or without changes in the sinuses. The atopic trait (RAST or immediate prick tests) or non-atopic asthma was not associated with any sinus abnormalities. In conclusion, if a close association between asthma and radiological disease of the sinuses exists (60% of cases), it does not seem that chronic sinusitis worsens bronchial obstruction or reduces sensitivity to beta-2-sympathomimetics. The changes in the sinus mucosa may be induced by the same aetiologic agent (allergic or non allergic) as asthma.

An example of interaction between environmental pollutants: modification of thiram toxicity to freshwater organisms by nitrites or nitrates in relation to nitrosamine synthesis.

Thiram, a dithiocarbamate fungicide, is known to evolve to dimethylnitrosamine (DMNA) when associated with nitrites. Conditions of appearance of that carcinogenic compound have been studied in short-term experiments by association of the fungicide, nitrates or nitrites, and species representative of freshwater biota. DMNA has been estimated by GLC equipped with a specific detector. Chlorella vulgaris can rapidly produce nitrites from nitrates and DMNA is obtained in presence of thiram. Daphnia magna can also synthesize DMNA but nitrites have to be added to the medium. Increased toxicity of thiram is observed. The same results are obtained on Cyprinus carpio and for a part on Brachydanio rerio. When the species are associated in a 15-day experimental food chain, and intoxicated algae feed the two other levels, no significant transfer is observed. Nevertheless, some DMNA hazard may exist for particular species exposed to thiram associated with nitrites or even nitrates if algae are present.

[Neuropathies caused by thalidomide]. / Neuropathies à la thalidomide.

Symptoms and signs in four patients with thalidomide-induced neuropathy developing during treatment of discoid lupus were limited for long period to distal paresthesiae with altered sensory conduction velocities. Semi-thin biopsy specimens of the distal sural nerve showed depopulation of myelinized fibers, mainly affecting those of large caliber, and signs of axonal degeneration. Study of dissociated fibers showed a high proportion of E fibers. Morphometry confirmed the axonal lesion. Ultrastructural examination demonstrated anomalies of axons of amyelinic fibers (vacuoles, lamellar figures) and of Schwann cells (stacked cytoplasmic prolongations), together with numerous collagen pockets, all non-specific lesions. The disease course was slow, with disappearance of sensory symptoms in a few weeks in 3 of the 4 cases and normal clinical findings in one of the four patients one year after cessation of treatment. Definite correlations between the dose administered and the severity of the neuropathy could not be established. The still poorly understood mechanism of action is discussed.

[Monilial granuloma treated successfully with ketoconazole over a period of 30 months]. / Granulome moniliasique traité avec succès par kétoconazole depuis 30 mois.

A case of monilial granuloma of 12 years duration is being successfully treated with ketoconazole administered orally in doses of 200 mg per day. Trush, onyxis and keratotic granulomatous lesions of the face and hands have dramatically regressed. In view of persistent anomalies of cellular immunity and complete deficiency in serum and salivary IgA, treatment with ketoconazole at the same dosage level has now been continuously pursued for 30 months without any evidence of toxicity.