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Object: Exploring mouse brains by rapid 3D-Diffusion Tensor Imaging (3D-DTI) of high spatial resolution (HSR) is challenging in vivo. Here we use the super resolution reconstruction (SRR) postprocessing method to demonstrate its performance on Microtubule-Associated-Protein6 Knock-Out (MAP6-KO) mice., Materials and Methods: Two spin-echo DTI were acquired (9.4T, CryoProbe RF-coil): (i)-multislice 2D-DTI, (echo-planar integrating reversed-gradient) acquired in vivo in the three orthogonal orientations (360 μm slice-thickness, 120 × 120 μm in-plane resolution, 56 min scan duration); used in SRR software to reconstruct SRR 3D-DTI with HSR in slice-plane (120 × 120 × 120 µm) and (ii)-microscopic 3D-DTI (µ-3D-DTI), (100 × 100 × 100 µm; 8 h 6 min) on fixed-brains ex vivo, that were removed after paramagnetic contrast-agent injection to accelerate scan acquisition using short repetition-times without NMR-signal sensitivity loss., Results: White-matter defects, quantified from both 3D-DTI fiber-tracking were found very similar. Indeed, as expected the fornix and cerebral-peduncle volume losses were - 39% and - 35% in vivo (SRR 3D-DTI) versus - 34% and - 32% ex vivo (µ-3D-DTI), respectively (p<0.001). This finding is robust since the µ-3D-DTI feasibility on MAP6-KO ex vivo was already validated by fluorescent-microscopy of cleared brains., Discussion: First performance of the SRR to generate rapid HSR 3D-DTI of mouse brains in vivo is demonstrated. The method is suitable in neurosciences for longitudinal studies to identify molecular and genetic abnormalities in mouse models that are of growing developments., (© 2023. The Author(s), under exclusive licence to European Society for Magnetic Resonance in Medicine and Biology (ESMRMB).)

Sunlight induces actinic keratosis, skin cancers and photoaging. Photoprotection is thus a major issue in public health to prevent the harmful effects of solar ultraviolet (UV) radiations. Recent data have shown that the visible (VIS) and infrared (IR) radiations can lead to skin damage by oxidative stress, suggesting that a balanced protection across the entire spectrum of sunlight is necessary to prevent cutaneous alterations. In this context, we developed a new generation of sunfilter called Phenylene Bis-Diphenyltriazine or TriAsorB (CAS N°55514-22-2). The aim of the present study was to assess the photoprotective efficacy of TriAsorB from UV to IR light. Spectrophotometric assays were performed to measure absorption and reflectance of TriAsorB in the different spectral ranges of sunlight: UV, VIS including blue light or high energy visible (HEV) and IR. DNA damage was evaluated using reconstructed human epidermis (RHE): 8-hydroxy-2'-deoxyguanosine (8OHdG) in response to HEV exposure, pyrimidine dimers (CPDs) and (6-4) photoproducts following solar-simulated radiation (SSR). TriAsorB is a broad spectrum UVB + UVA filter including long UVA. Interestingly, it also absorbs VIS radiations, especially in the HEV region. These radiations are also reflected. Protection in the IR spectral range is weak. Furthermore, the sunfilter specifically protects the skin against the oxidative lesions 8OHdG induced by HEV and prevents SSR-induced DNA damage. Thus, TriAsorB is an innovative sunfilter that might be used in sun care products for skin photoprotection from UV to VIS radiations. Finally, it prevents sunlight genotoxicity and protected the skin against solar radiations, especially blue light., (© 2021. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations' mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.

Hydrogenases are metalloenzymes that catalyse both H 2 evolution and uptake. They are gas-processing enzymes with deeply buried active sites, so the gases diffuse through channels that connect the active site to the protein surface. The [NiFeSe] hydrogenases are a special class of hydrogenases containing a selenocysteine as a nickel ligand; they are more catalytically active and less O 2 -sensitive than standard [NiFe] hydrogenases. Characterisation of the channel system of hydrogenases is important to understand how the inhibitor oxygen reaches the active site to cause oxidative damage. To this end, crystals of Desulfovibrio vulgaris Hildenborough [NiFeSe] hydrogenase were pressurized with krypton and oxygen, and a method for tracking labile O 2 molecules was developed, for mapping a hydrophobic channel system similar to that of the [NiFe] enzymes as the major route for gas diffusion.

During the course of evolution, variations of a protein sequence is an ongoing phenomenon however limited by the need to maintain its structural and functional integrity. Deciphering the evolutionary path of a protein is thus of fundamental interest. With the development of new methods to visualize high dimension spaces and the improvement of phylogenetic analysis tools, it is possible to study the evolutionary trajectories of proteins in the sequence space. Using the data-driven high-dimensional scaling method, we show that it is possible to predict and represent potential evolutionary trajectories by representing phylogenetic trees into a 3D projection of the sequence space. With the case of the aminodeoxychorismate synthase, an enzyme involved in folate synthesis, we show that this representation raises interesting questions about the complexity of the evolution of a given biological function, in particular concerning its capacity to explore the sequence space.

The article "Iron-sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding", written by J. Pérard, Sandrine Ollagnier de Choudens was originally published electronically on the publisher's internet portal (currently SpringerLink) 26 December, 2017 without open access.

Iron-sulfur clusters (Fe-S) are amongst the most ancient and versatile inorganic cofactors in nature which are used by proteins for fundamental biological processes. Multiprotein machineries (NIF, ISC, SUF) exist for Fe-S cluster biogenesis which are mainly conserved from bacteria to human. SUF system (sufABCDSE operon) plays a general role in many bacteria under conditions of iron limitation or oxidative stress. In this mini-review, we will summarize the current understanding of the molecular mechanism of Fe-S biogenesis by SUF. The advances in our understanding of the molecular aspects of SUF originate from biochemical, biophysical and recent structural studies. Combined with recent in vivo experiments, the understanding of the Fe-S biogenesis mechanism considerably moved forward.

Surgery precedes both radiotherapy and chemotherapy as the first-line therapy for glioma. However, despite multimodal treatment, most glioma patients die from local recurrence in the resection margin. Glioma surgery is inherently lesional, and the response of brain tissue to surgery includes hemostasis, angiogenesis, reactive gliosis and inflammation. Unfortunately, these processes are also associated with tumorigenic side-effects. An increasing amount of evidence indicates that the response to a surgery-related brain injury is hijacked by residual glioma cells and participates in the local regeneration of tumor tissues at the resection margin. Inducing therapeutic hypothermia in the brain has long been used to treat the secondary damage, such as neuroinflammation and edema, that are caused by accidental traumatic brain injuries. There is compelling evidence to suggest that inducing therapeutic hypothermia at the resection margin would delay the local recurrence of glioma by (i) limiting cell proliferation, (ii) disrupting the pathological connection between inflammation and glioma recurrence, and (iii) limiting the consequences of the functional heterogeneity and complexity inherent to the tumor ecosystem. While the global whole-body cooling methods that are currently used to treat stroke in clinical practice may not adequately treat the resection margin, the future lies in implantable focal microcooling devices similar to those under development for the treatment of epilepsy. Preclinical and clinical strategies to evaluate focal hypothermia must be implemented to prevent glioma recurrence in the resection margin. Placing the resection margin in a state of hibernation may potentially provide such a long-awaited therapeutic breakthrough.

Idiopathic non-histaminergic acquired angioedema (InH-AAE) is a rare disease characterized by AE resistant to antihistamines and a chronic course. We report five new cases of InH-AAE (two women and three men) with a rapid and dramatic response to the anti-immunoglobulin-E antibody omalizumab. In our literature review, we found 13 other relevant cases with a good response to this treatment. Overall, in 6 out of 18 patients, the doses of omalizumab required to prevent recurrences of attacks were higher than the licensed dose for chronic urticaria. No significant adverse effects have been reported.

The enhancement of the performance of advanced nitride-based optoelectronic devices requires the fine tuning of their composition, which has to be determined with a high accuracy and at the nanometer scale. For that purpose, we have evaluated and compared energy dispersive X-ray spectroscopy (EDX) in a scanning transmission electron microscope (STEM) and atom probe tomography (APT) in terms of composition analysis of AlGaN/GaN multilayers. Both techniques give comparable results with a composition accuracy better than 0.6 % even for layers as thin as 3 nm. In case of EDX, we show the relevance of correcting the X-ray absorption by simultaneous determination of the mass thickness and chemical composition at each point of the analysis. Limitations of both techniques are discussed when applied to specimens with different geometries or compositions.

Nannochloropsis is an eukaryotic alga of the phylum Heterokonta, originating from a secondary endosymbiotic event. In this work, we investigated how the photosynthetic apparatus responds to growth in different light regimes in Nannochloropsis gaditana. We found that intense illumination induces the decrease of both photosystem I and II contents and their respective antenna sizes. Cells grown in high light showed a larger capacity for electron transport, with enhanced cyclic electron transport around photosystem I, contributing to photoprotection from excess illumination. Even when exposed to excess light intensities for several days, N. gaditana cells did not activate constitutive responses such as nonphotochemical quenching and the xanthophyll cycle. These photoprotection mechanisms in N. gaditana thus play a role in acclimation to fast changes in illumination within a time range of minutes, while regulation of the electron flow capacity represents a long-term response to prolonged exposure to excess light.

Radical SAM enzymes generally contain a [4Fe-4S](2+/1+) (RS cluster) cluster bound to the protein via the three cysteines of a canonical motif CxxxCxxC. The non-cysteinyl iron is used to coordinate SAM via its amino-carboxylate moiety. The coordination-induced proximity between the cluster acting as an electron donor and the adenosyl-sulfonium bond of SAM allows for the homolytic cleavage of the latter leading to the formation of the reactive 5'-deoxyadenosyl radical used for substrate activation. Most of the structures of Radical SAM enzymes have been obtained in the presence of SAM, and therefore, little is known about the situation when SAM is not present. In this report, we show that RimO, a methylthiotransferase belonging to the radical SAM superfamily, binds a Tris molecule in the absence of SAM leading to specific spectroscopic signatures both in Mössbauer and pulsed EPR spectroscopies. These data provide a cautionary note for researchers who work with coordinative unsaturated iron sulfur clusters.

Airborne particles are known to cause illness and to influence meteorological phenomena. It is therefore important to monitor their concentrations and to identify them. A challenge is to collect micro and nanoparticles, microorganisms as well as toxic molecules with a device as simple and small as possible to be used easily and everywhere. Electrostatic precipitation is an efficient method to collect all kinds of airborne particles. Furthermore, this method can be miniaturized. A portable, silent, and autonomous air sampler based on this technology is therefore being developed with the final objective to collect very efficiently airborne pathogens such as supermicron bacteria but also submicron viruses. Particles are collected on a dry surface so they may be concentrated afterwards in a small amount of liquid medium to be analyzed. It is shown that nearly 98 % of airborne particles from 10 nm to 3 μm are collected.

Chemical genetics has emerged as a powerful approach to dissect biological processes, based on the utilization of small molecules disturbing the function of specific target proteins. By analogy with classical genetics, 'reverse chemical genetics' refers to the utilization of drugs acting on a known target, enabling its functional characterization at the levels of the cells, tissues and organisms. Likewise, 'direct chemical genetics' refers to the utilization of a drug of unknown mode of action, but triggering a phenotype of interest. In that case, one has to identify the target(s) possibly blocked (or possibly activated) by the small molecule. This chapter illustrates both approaches, like the analysis of the elongation of fatty acids, the biosynthesis of galactoglycerolipids or the catabolism of phosphoglycerolipids by reverse chemical genetics or the study of the membrane glycerolipid remodeling triggered upon phosphate starvation, by direct chemical genetics.

Introduction: Bradykinin-mediated angioedema (AE) is a rare side effect of some medications, including angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB). In France, side-effects to treatments are reported to the national pharmacovigilance database., Methods: The national MedDRA database was searched using the term "angioedema". Patients were included if they met the clinical criteria corresponding to bradykinin-mediated AE, if their C1-inhibitor levels were normal, and if they were treated with an ACEi or an ARB., Results: 7998 cases of AE were reported between 1994 and 2013. Among these, 112 met the criteria for bradykinin-mediated AE with normal C1-inhibitor levels. On the 112 drug-AE, patients were treated with an ARB in 21% of cases (24 patients), or an ACEi in 77% of cases (88 patients), in combination with another treatment in 17 cases (mTORi for 3 patients, iDPP-4 for 1 patient, hormonal treatment for 7 patients). ENT involvement was reported in 90% of cases (tongue: 48.2%, larynx: 23.2%). The median duration of treatment before the first attack was 720 days, and the mean duration of attacks was 36.6 h. Forty-one percent (19/46) of patients relapsed after discontinuing treatment., Conclusion: Angioedema triggered by medication blocking the renin/angiotensin system is rare but potentially severe, with a high risk of recurrence despite cessation of the causative drug.

The transcription factor NF-κB plays a key role in numerous physiological processes such as inflammation, immunity, cell proliferation or control of cell death. Its activation is tightly controlled by a kinase complex, IκB kinase (IKK), composed of three core proteins: IKK1/IKKα, IKK2/IKKβ and NEMO/IKKγ. The first two are structurally related kinases whereas the third one is a regulatory subunit exhibiting affinity for upstream activators modified by polyubiquitin chains. Over the years, several inherited diseases caused by mutations of each of the three subunits of IKK have been identified in humans together with diseases caused by mutations of several of its substrates. They are associated with very specific and complex phenotypes involving a broad range of abnormalities such as impaired innate and acquired immune response, perturbed skin development and defects of the central nervous system. Here, we summarize the diverse clinical, cellular and molecular manifestations of IKK-related genetic diseases and show that studying patient-related mutations affecting the IKK subunits and some of their substrates offers the opportunity to understand the various functions of NF-κB in humans, complementing studies performed with mouse models. This analysis also provides glimpses about putative functions of IKK subunits that may be NF-κB-independent.

The 5-methylcytosine derivative 5-hydroxymethylcytosine (5hmCyt), which is generated via enzymatic oxidation, is sometimes referred to as the sixth nucleobase due to its widespread presence in the DNA of brain and embryonic stem cells. In this study, we used density functional based methods and reactivity indices from conceptual DFT to explore the mechanism and key features of the hydrolytic deamination of 5hmCyt. The data obtained are used to compare and contrast this deamination reaction with those of other cytosine derivatives. The deamination process for 5hmCyt is similar to the corresponding processes for other unsaturated derivatives in that the amino form is the reactive one and water addition is the rate-limiting step. However, several differences due to the rotameric asymmetry of the current system are also noted.

The biosynthesis of the organometallic H cluster of [Fe-Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe-S protein with GTPase activity. The [4Fe-4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe-4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe-4S](2+/+) clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe-4S] cluster and the non-imidazole-bound [4Fe-4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe-4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.

In this work, the third derivative of the energy with respect to the number of electrons, the so-called hyper-hardness, is investigated to assess whether this quantity has a chemical meaning. To achieve this goal a new working expression for hyper-hardness is developed and analyzed. It transpired from this analysis that hyper-hardness, just like hardness, can measure the reactivity or the stability of electron systems. Interestingly, positive values of hyper-hardness point to quite stable species such as noble gases and molecules. On the other hand, radicals almost always display large negative values of hyper-hardness.

Identifiable causes of fetal growth restriction (FGR) account for 30 % of cases, but the remainders are idiopathic and are frequently associated with placental dysfunction. We have shown that the angiogenic factor endocrine gland-derived VEGF (EG-VEGF) and its receptors, prokineticin receptor 1 (PROKR1) and 2, (1) are abundantly expressed in human placenta, (2) are up-regulated by hypoxia, (3) control trophoblast invasion, and that EG-VEGF circulating levels are the highest during the first trimester of pregnancy, the period of important placental growth. These findings suggest that EG-VEGF/PROKR1 and 2 might be involved in normal and FGR placental development. To test this hypothesis, we used placental explants, primary trophoblast cultures, and placental and serum samples collected from FGR and age-matched control women. Our results show that (1) EG-VEGF increases trophoblast proliferation ([(3)H]-thymidine incorporation and Ki67-staining) via the homeobox-gene, HLX (2) the proliferative effect involves PROKR1 but not PROKR2, (3) EG-VEGF does not affect syncytium formation (measurement of syncytin 1 and 2 and β hCG production) (4) EG-VEGF increases the vascularization of the placental villi and insures their survival, (5) EG-VEGF, PROKR1, and PROKR2 mRNA and protein levels are significantly elevated in FGR placentas, and (6) EG-VEGF circulating levels are significantly higher in FGR patients. Altogether, our results identify EG-VEGF as a new placental growth factor acting during the first trimester of pregnancy, established its mechanism of action, and provide evidence for its deregulation in FGR. We propose that EG-VEGF/PROKR1 and 2 increases occur in FGR as a compensatory mechanism to insure proper pregnancy progress.

We address two aspects of general interest for the chemical synthesis of colloidal semiconductor nanocrystals: (1) the rational design of the synthesis protocol aiming at the optimization of the reaction parameters in a minimum number of experiments; (2) the transfer of the procedure to the gram scale, while maintaining a low size distribution and maximizing the reaction yield. Concerning the first point, the design-of-experiment (DOE) method has been applied to the synthesis of colloidal CdSe nanocrystals. We demonstrate that 16 experiments, analyzed by means of a Taguchi L16 table, are sufficient to optimize the reaction parameters for controlling the mean size of the nanocrystals in a large range while keeping the size distribution narrow (5-10%). The DOE method strongly reduces the number of experiments necessary for the optimization as compared to trial-and-error approaches. Furthermore, the Taguchi table analysis reveals the degree of influence of each reaction parameter investigated (e.g., the nature and concentration of reagents, the solvent, the reaction temperature) and indicates the interactions between them. On the basis of these results, the synthesis has been scaled up by a factor of 20. Using a 2-L batch reactor combined with a high-throughput peristaltic pump, different-sized samples of CdSe nanocrystals with yields of 2-3 g per synthesis have been produced without sacrificing the narrow size distribution. In a similar setup, the gram-scale synthesis of CdSe/CdS/ZnS core/shell/shell nanocrystals exhibiting a fluorescence quantum yield of 81% and excellent resistance of the photoluminescence in presence of a fluorescent quencher (aromatic thiol) has been achieved.PACS: 81.20.Ka, 81.07.Bc, 78.67.Bf.

Numerous luminophores may be encapsulated into silica nanoparticles (< 100 nm) using the reverse microemulsion process. Nevertheless, the behaviour and effect of such luminescent molecules appear to have been much less studied and may possibly prevent the encapsulation process from occurring. Such nanospheres represent attractive nanoplatforms for the development of biotargeted biocompatible luminescent tracers. Physical and chemical properties of the encapsulated molecules may be affected by the nanomatrix. This study examines the synthesis of different types of dispersed silica nanoparticles, the ability of the selected luminophores towards incorporation into the silica matrix of those nanoobjects as well as the photophysical properties of the produced dye-doped silica nanoparticles. The nanoparticles present mean diameters between 40 and 60 nm as shown by TEM analysis. Mainly, the photophysical characteristics of the dyes are retained upon their encapsulation into the silica matrix, leading to fluorescent silica nanoparticles. This feature article surveys recent research progress on the fabrication strategies of these dye-doped silica nanoparticles.

Strict and facultative anaerobes depend on a class III ribonucleotide reductase for their growth. These enzymes are the sole cellular catalysts for de novo biosynthesis of the deoxyribonucleotides needed for DNA chain elongation and repair. In its active form, the class III ribonucleotide reductase from Escherichia coli contains a free radical located on the G681 residue which is essential for the activation of the ribonucleotide substrate toward its reduction. The 3D structure of the homologous enzyme from bacteriophage T4 has revealed the presence of a metal center bound to four conserved cysteine residues. In this report we identify the metal of the E. coli enzyme as Zn. We show that the presence of Zn in this site protects the protein from proteolysis and prevents the formation of disulfide bridges within it. Finally, we show with the fully Zn-loaded reductase that thioredoxin or small thiols are dispensable for the formation of the glycyl radical. However, they are necessary for obtaining high turnover numbers, suggesting that they intervene in radical transfer steps subsequent to the formation of the glycyl radical.

The survey is aimed at critically reviewing information on the UVA-mediated oxidative reactions to cellular components with emphasis on DNA as the result of mostly photosensitized pathways. It appears clearly that UVA radiation is relatively much more efficient than UVB photons in inducing oxidative processes. The main UVA-induced oxidative degradation pathways of DNA are reported and discussed mechanistically. They are mostly rationalized in terms of a major contribution of singlet molecular oxygen ((1)O(2)) and to a lesser extent of hydroxyl radical ( OH), that in the latter case originates from Fenton-type reactions. This leads to the predominant formation of 8-oxo-7,8-dihydroguanine together with smaller amounts of oxidized pyrimidine bases and DNA strand breaks in UVA-irradiated cells.

An inventory of unique local protein folds around Fe-S clusters has been derived from the analysis of protein structure databases. Nearly 50 such folds have been identified, and over 90% of them harbor low-potential [2Fe-2S](2+,+) or [4Fe-4S](2+,+) clusters. In contrast, high-potential Fe-S clusters, notwithstanding their structural diversity, occur in only three different protein folds. These observations suggest that the extant population of Fe-S protein folds has to a large extent been shaped in the reducing iron- and sulfur-rich environment that is believed to have predominated on this planet until approximately two billion years ago. High-potential active sites are then surmised to be rarer because they emerged later, in a more oxidizing biosphere, in conditions where iron and sulfide had become poorly available, Fe-S clusters were less stable, and in addition faced competition from heme iron and copper active sites. Among the low-potential Fe-S active sites, protein folds hosting [4Fe-4S](2+,+) clusters outnumber those with [2Fe-2S](2+,+) ones by a factor of 3 at least. This is in keeping with the higher chemical stability and versatility of the tetranuclear clusters, compared with the binuclear ones. It is therefore suggested that, at least while novel Fe-S sites are evolving within proteins, the intrinsic chemical stability of the inorganic moiety may be more important than the stabilizing effect of the polypeptide chain. The discovery rate of novel Fe-S-containing protein folds underwent a sharp increase around 1995, and has remained stable to this day. The current trend suggests that the mapping of the Fe-S fold space is not near completion, in agreement with predictions made for protein folds in general. Altogether, the data collected and analyzed here suggest that the extant structural landscape of Fe-S proteins has been shaped to a large extent by primeval geochemical conditions on one hand, and iron-sulfur chemistry on the other.

Most hydrogenases (H2ases), the enzymes that produce or oxidize dihydrogen, possess dimetallic active sites and belong to either one of two phylogenetically distinct classes, the [NiFe] and the [FeFe] H2ases. These families of H2ases share a number of similarities regarding active site structure and reaction mechanism, as a result of convergent evolution. They are otherwise alien to each other, in particular with respect to protein sequence and structure, maturation mechanisms, and distribution among the realms of life. One of the interesting features of [FeFe] H2ases is their occurrence in anaerobic bacteria, anaerobic protists, and mitochondriate eukaryotes. They thus have the potential to report on important evolutionary events, including transitions from the prokaryote to the eukaryote lifestyle. Genome sequences yield a variety of [FeFe] H2ase sequences that have been implemented to shed light on the evolution of these proteins and their host organisms.

Chloroplasts are bounded by a pair of outer membranes, the envelope, that is the only permanent membrane structure of the different types of plastids. Chloroplasts have had a long and complex evolutionary past and integration of the envelope membranes in cellular functions is the result of this evolution. Plastid envelope membranes contain a wide diversity of lipids and terpenoid compounds serving numerous biochemical functions and the flexibility of their biosynthetic pathways allow plants to adapt to fluctuating environmental conditions (for instance phosphate deprivation). A large body of knowledge has been generated by proteomic studies targeted to envelope membranes, thus revealing an unexpected complexity of this membrane system. For instance, new transport systems for metabolites and ions have been identified in envelope membranes and new routes for the import of chloroplast-specific proteins have been identified. The picture emerging from our present understanding of plastid envelope membranes is that of a key player in plastid biogenesis and the co-ordinated gene expression of plastid-specific protein (owing to chlorophyll precursors), of a major hub for integration of metabolic and ionic networks in cell metabolism, of a flexible system that can divide, produce dynamic extensions and interact with other cell constituents. Envelope membranes are indeed one of the most complex and dynamic system within a plant cell. In this review, we present an overview of envelope constituents together with recent insights into the major functions fulfilled by envelope membranes and their dynamics within plant cells.

The catalase from Proteus mirabilis peroxide-resistant bacteria is one of the most efficient heme-containing catalases. It forms a relatively stable compound II. We were able to prepare samples of compound II from P. mirabilis catalase enriched in (57)Fe and to study them by spectroscopic methods. Two different forms of compound II, namely, low-pH compound II (LpH II) and high-pH compound II (HpH II), have been characterized by Mössbauer, extended X-ray absorption fine structure (EXAFS) and UV-vis absorption spectroscopies. The proportions of the two forms are pH-dependent and the pH conversion between HpH II and LpH II is irreversible. Considering (1) the Mössbauer parameters evaluated for four related models by density functional theory methods, (2) the existence of two different Fe-O(ferryl) bond lengths (1.80 and 1.66 A) compatible with our EXAFS data and (3) the pH dependence of the alpha band to beta band intensity ratio in the absorption spectra, we attribute the LpH II compound to a protonated ferryl Fe(IV)-OH complex (Fe-O approximately 1.80 A), whereas the HpH II compound corresponds to the classic ferryl Fe(IV)=O complex (Fe=O approximately 1.66 A). The large quadrupole splitting value of LpH II (measured 2.29 mm s(-1) vs. computed 2.15 mm s(-1)) compared with that of HpH II (measured 1.47 mm s(-1) vs. computed 1.46 mm s(-1)) reflects the protonation of the ferryl group. The relevancy and involvement of such (Fe(IV)=O/Fe(IV)-OH) species in the reactivity of catalase, peroxidase and chloroperoxidase are discussed.

This last decade, many efforts were undertaken to understand how coenzymes, including vitamins, are synthesized in plants. Surprisingly, these metabolic pathways were often "quartered" between different compartments of the plant cell. Among these compartments, mitochondria often appear to have a key role, catalyzing one or several steps in these pathways. In the present review we will illustrate these new and important biosynthetic functions found in plant mitochondria by describing the most recent findings about the synthesis of two vitamins (folate and biotin) and one non-vitamin coenzyme (lipoate). The complexity of these metabolic routes raise intriguing questions, such as how the intermediate metabolites and the end-product coenzymes are exchanged between the various cellular territories, or what are the physiological reasons, if any, for such compartmentalization.

Exposure of DNA to far-UV radiation leads to the formation of several types of dimeric lesions, including cyclobutane dimers and (6-4) photoproducts. In order to gain insights into the main parameters driving DNA photochemistry, the effect of ionic strength on the yield of formation of these photoproducts was investigated in UVC-irradiated samples of isolated genomic DNA. The main consequence of lowering the ionic strength was a decrease in the UVC-induced formation of thymine-cytosine and cytosine-cytosine photoproducts. The reactivity of thymine-thymine and cytosine-thymine doublets was hardly affected. Evidence was obtained against a major role played by duplex denaturation in these observations. A more likely explanation is a change in the DNA structure as the result of a larger extent of protonation at low counter-ions concentration.

Biosynthesis of iron-sulfur clusters is a cellular process which depends on complex protein machineries. Escherichia coli contains two such biosynthetic systems, ISC and SUF. In this review article we specifically make a presentation of the various Suf proteins and discuss the molecular mechanisms by which these proteins work together to assemble Fe and S atoms within a scaffold and to transfer the resulting cluster to target proteins.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.

Knocking out the regulatory beta subunit of protein kinase CK2 in mice leads to early embryonic lethality. Heterozygous CK2beta (CK2beta+/-) knockout mice do not show an obvious phenotype. However, the number of heterozygous offsprings from CK2B+/- inter-crossings is lower than expected, meaning that some heterozygous embryos do not survive. Interestingly, CK2beta+/- ES (Embryonic Stem) cells express a considerably lower level of CK2beta than wild-type ES cells, whereas the level of CK2beta in organs from heterozygous adult mice does not significantly differ from those of wild-type mice. The data suggest a compensatory mechanism that adjusts CK2beta levels during development in the majority of, but not in all, cases (Mol Cell Biol 23: 908-915, 2003). In order to find an explanation for the gene dosage effect observed for heterozygous offsprings, we analysed embryos at mid-gestation (E10.5) as well as wild-type and CK2beta+/- ES cells for differences in growth rate and response to different stress agents. Analysis of E10.5 embryos generated from heterozygous matings revealed about 20% of smaller retarded CK2beta+/- embryos. No correlation between CK2beta levels in normal looking and retarded CK2beta+/- embryos were found. However, a different post-translational form of CK2beta protein has been detected in these retarded embryos. Cellular parameters such as growth rate and G1-, G2-checkpoints in ES cells were identical in both wild-type and CK2beta+/- cells. When ES cells were injected to induce differentiated teratocarcinoma in syngenic mice, the size of the tumours correlated with the level of CK2beta.

Triadin is a protein first identified as a member of the muscle calcium release complex, involved in calcium release for muscle contraction. However, its precise function in this complex is still undefined. Recently, triadin has been shown to be a multi-protein family, with different distribution of the various splice variants within the sarcoplasmic reticulum, raising the possibility of multiple functions for this family of polypeptides. Such functions may include involvement in excitation-contraction coupling, in triad targeting, in structural function or in muscle differentiation. The putative role(s) of triadin(s) will be discussed here.

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Deltaa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. (15)N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.

Far-UV irradiation of DNA leads to the formation of several types of dimeric lesions between adjacent pyrimidine bases including cyclobutane dimers, (6-4) photoproducts and Dewar valence isomers In the dry state, an additional specific thymine lesion, the spore photoproduct, is produced. We designed an HPLC-tandem mass spectrometry assay for the detection of the latter lesion. This technique that does not require radio-labelling of DNA allowed the simultaneous quantification of the spore photoproduct and other pyrimidine dimeric photoproducts. Using this approach, the complete distribution of bipyrimidine lesions within UVC-irradiated dry DNA was determined.

Flavocytochrome b558 is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O2 into the superoxide anion O2 in phagocytic cells. Flavocytochrome b558 is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox forphagocyte oxidase) (beta subunit) and a small protein p22phox (alpha subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b558 which becomes activated and generates O2-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O2- and the derived metabolites H2O2 and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b558. The p22phox subunit serves as a docking site for the cytoso lic phox proteins. This review provides an overview of current knowledge on the structural organization of the O2(-)-generating flavocytochrome b558, its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O2(-)-generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed.

The two diastereoisomers of 6-hydroxy-5,6-dihydro-2'-deoxyuridine (dUrd hydrates) are produced upon deamination of the related 2'-deoxycytidine derivatives. Liquid chromatography coupled to tandem mass spectrometry was used to quantify dUrd hydrates. Their rate constant of formation within UVC-irradiated solutions of dCyd was first determined. Their formation was also shown to occur within solutions of either dUrd or dCyd exposed to gamma-rays in the absence of oxygen. dUrd hydrates were then quantified within isolated and cellular DNA exposed to UVC light. In both cases, their yield of formation was found to be, at least, 2 orders of magnitude lower than the overall yield of bipyrimidine photoproducts such as cyclobutane dimers and (6-4) photoproducts.

The magnetic properties of the reduced catalase from Lactobacillus plantarum have been studied for the active enzyme and its fluoride complex through variable field/variable temperature magnetization measurements. The magnetic exchange interaction deduced from these experiments [fluoride complex: - J=1.3(1) cm(-1); active enzyme: - J=5.6(5) cm(-1); H=-2 J S(1) S(2)] are similar to those presently obtained in a re-analysis of the data for the corresponding forms of the Thermus thermophilus enzyme (previously published in 1997, Angew Chem Int Ed Engl 36:1626-1628): phosphate complex: - J=2.1(2) cm(-1); active enzyme - J=5.0(3) cm(-1). These results concur to a unified picture for the two enzymes, consistent with the presence of a hydroxide bridge in the reduced active catalases and its replacement by an aqua bridge in the anion-inhibited enzymes as the main mediators of the magnetic exchange.

A three-dimensional MQ-TROSY-HCN pulse sequence is presented which provides intra-base and sugar-to-base correlations for 13C, 15N labeled nucleic acids (RNA, DNA). The experiment simultaneously exploits the favorable relaxation properties of 1H-13C multiple quantum coherence for sugar carbons and of 13C TROSY-type spin evolution for base carbons. MQ-TROSY-HCN thus combines the advantages of MQ-HCN for sugar-to-base and TROSY-HCN for intra-base correlations in a single experiment. In addition, two slightly different implementations of the MQ-TROSY-HCN experiment ensure optimal performance for small and larger oligonucleotides, respectively. The advantages of the MQ-TROSY-HCN experiment compared to the best previous implementations of HCN are demonstrated for a 33 nucleotide RNA aptamer.

Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions. Microtubules are major organizers of the cell interior and are vitally involved in motility events such as chromosome migration during cell division. To fulfill their physiological function, microtubule arrays have to undergo dramatic changes in their spatial arrangement, and this depends to a large extent on the complex and special dynamic properties of the individual polymers. In this review we first describe the intrinsic dynamic properties of microtubules assembled in vitro from purified tubulin and examine the relationships between these properties and microtubule functions. Subsequent sections concern microtubule dynamics in vivo, their similarity and differences with microtubule dynamics in vitro, and the nature of the cellular regulators which act on microtubule assemblies in physiological conditions.

We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2alpha or GFP-CK2beta showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance.

Complex I [NADH-ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB-NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.

The semi-classical electron transfer theory has been very successful in describing reactions occurring in biological systems, but the relevant parameters in the case of iron-sulfur proteins remain unknown. The recent discovery that 2[4Fe-4S] proteins homologous to Chromatium vinosum ferredoxin contain clusters with different reduction potentials now gives the opportunity to study the dependence of the intramolecular electron transfer rate between these clusters as a function of the driving force. This work shows how decreasing the reduction potential difference between the clusters by site-directed mutagenesis of C. vinosum ferredoxin modifies the rate of electron hopping between the two redox sites of the protein by measuring the line broadening of selected 1H NMR signals. Beside the shifts of the reduction potentials, no signs of large structural changes or of significant alterations of the intrinsic kinetic parameters among the different variants of C. vinosum ferredoxin have been found. A reorganization energy of less than 0.5 eV was deduced from the dependence of the electron transfer rates with the reduction potential difference. This small value is associated with a weak electronic coupling between the two closely spaced clusters. This set of parameters, determined for the first time in an iron-sulfur protein, may help to explain how efficient vectorial electron transfer occurs with a small driving force in the many enzymatic systems containing a 2[4Fe-4S] domain.

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear 13C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any 13C-edited multidimensional NMR experiment. As an example we have chosen the 13C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.

Knowledge of the native disulphide bridge topology allows the introduction of conformational restraints between remote parts of the peptide chain. This information is therefore of great importance for the successful determination of the three-dimensional structure of cysteine-rich proteins by NMR spectroscopy. In this paper we investigate the limitations of using ambiguous intersulphur restraints [Nilges, M. (1995) J. Mol. Biol., 245, 645-660] associated with NMR experimental information to determine the native disulphide bridge pattern. Using these restraints in a simulated annealing protocol we have determined the correct topology of numerous examples, including a protein with seven disulphide bridges (phospholipase A2) and a protein in which 25% of the total number of residues are cysteines (mu-conotoxin GIIIB). We have also characterised the behaviour of the method when only limited experimental data is available, and find that the proposed protocol permits disulphide bridge determination even with a small number of restraints (around 5 NOEs--including a long-range restraint--per residue). In addition, we have shown that under these conditions the use of a reduced penalty function allows the identification of misassigned NOE restraints. These results indicate that the use of ambiguous intersulphur distances with the proposed simulated annealing protocol is a general method for the determination of disulphide bridge topology, particularly interesting in the first steps of NMR study of cysteine-rich proteins. Comparison with previously proposed protocols indicates that the presented method is more reliable and the interpretation of results is straightforward.

The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles.

We have characterized several subdomains of the beta subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2beta subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane. The C-terminal domain of the CK2beta subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the beta subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.