The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Synthesis, resolution, and determination of absolute configuration of protected α-ethynylphenylalanine enantiomers.

Racemic-protected α-ethynylphenylalanine was synthesized from DL-2-benzylserine using α-benzylserinal as key intermediate and was successfully resolved by HPLC on a chiral stationary phase at a semipreparative scale. The absolute configuration of both enantiomers was determined by vibrational circular dichroism.

Respective impact of climate and fisheries on the growth of an albatross population.

Climate and human activities such as fisheries impact many animal species. However, the demographic processes through which the population vital rates are affected, and the sensitivity of their growth rates, are poorly understood. The Black-browed Albatross, Thalassarche melanophrys, is a long-lived threatened seabird species. Previous studies have shown that the adult survival and breeding success of the population breeding at Kerguelen are affected by sea surface temperature anomalies (SSTA) during both the breeding and the nonbreeding season, and by tuna long-lining in Tasmanian waters through bycatch mortality. Here, using long-term demographic data from a Black-browed Albatross colony monitored for 26 years at Kerguelen, we estimate all demographic parameters from early to adult stages of the life cycle in order to build a fully parameterized population model and predict population growth rates under several scenarios of climate and fishing effort. The observed population growth rate (1.003) indicates that the population was stable or slightly increasing, and our population model gives a close estimate of 1.008. Population growth rate is more sensitive to survival of experienced breeders and accordingly to a change in SSTA during incubation and to tuna long-lining effort (both affecting survival of experienced breeders) than to other demographic parameters/environmental covariates. The population stability results from multiple factors and complex relationships between demographic parameters and environmental conditions, and therefore population equilibrium is precarious. If fishing effort remains stable at its current level and positive SSTA increase, or inversely if fishing effort decreases and SSTA remain similar to present values, then the population would increase. However, if fishing effort increases by 20% (i.e., to 40 million hooks) on the wintering grounds, without any change in SSTA, then the population would decrease at 0.9% per year. If fishing effort stops, the population would increase at 3.5% per year, suggesting that bycatch mortality probably currently limits the Black-browed Albatross population at Kerguelen. Our study shows how this type of model could be useful to predict trajectories of top predator populations, and eventually lower trophic web levels, in relation to climatic projections and future human activities. We highlight the need to reinforce mitigation measures.

Risk assessment of substances that are both genotoxic and carcinogenic report of an International Conference organized by EFSA and WHO with support of ILSI Europe.

The European Food Safety Authority (EFSA) and the World Health Organization (WHO), with the support of the International Life Sciences Institute, European Branch (ILSI Europe), organized an international conference on 16-18 November 2005 to discuss how regulatory and advisory bodies evaluate the potential risks of the presence in food of substances that are both genotoxic and carcinogenic. The objectives of the conference were to discuss the possible approaches for risk assessment of such substances, how the approaches may be interpreted and whether they meet the needs of risk managers. ALARA (as low as reasonably achievable) provides advice based solely on hazard identification and does not take into account either potency or human exposure. The use of quantitative low-dose extrapolation of dose-response data from an animal bioassay raises numerous scientific uncertainties related to the selection of mathematical models and extrapolation down to levels of human exposure. There was consensus that the margin of exposure (MOE) was the preferred approach because it is based on the available animal dose-response data, without extrapolation, and on human exposures. The MOE can be used for prioritisation of risk management actions but the conference recognised that it is difficult to interpret it in terms of health risk.

Full stereochemical understanding in a new (2R,3R,4R)-4-hydroxyisoleucine synthesis.

We present the crystal and molecular structures of 2,3,6,7,8,8a-hexahydro-6,8-methano-7,7,8a-trimethyl-3-(1-methyl-2-oxopropylidene)-5H-1,4-benzoxazin-2-one, C16H21NO3, (III), and 2,3,6,7,8,8a-hexahydro-3-(2-hydroxy-1-methylpropyl)-6,8-methano-7,7,8a-trimethyl-5H-1,4-benzoxazin-2-one, C16H25NO3, (V). These compounds are two of the four key intermediates in our synthetic route to (2R,3R,4R)-4-hydroxyisoleucine. The two structures provide a full understanding of the stereochemistry in successive steps. This synthesis was based on a new optically pure chiral oxazinone auxiliary derived from (1R,2R,5R)-2-hydroxypinan-3-one.

Prediction of basal metabolism from organ size in the rat: relationship to strain, feeding, age, and obesity.

Use of the weight of various organs and tissues together with their specific metabolic activity for prediction of basal metabolism (BM) seems to be promising. In this study we compared the use of this method with those based on simple or multiple regression analyses. We observed that 97.4% of differences in BM in a group of nine adult male Wistar rats weighing 273--517 g could be accounted for by changes in tissue and organ weights. BM measured in lean Zucker and Sprague-Dawley rats did not diverge from the prediction of the model by >1.6%. According to the organ-based model as well as multiple regression analyses, but not simple regression analyses, BM was increased 18--21% in young rats, decreased 6--7% in food restricted/refed rats, and decreased 19--21% in aged rats. Only with obese rats did the predictions of the two methods diverge. The main reason for this discrepancy seems to be the way adipose tissue size and metabolism are taken into account.

Chemoenzymatic synthesis of surfactants from carbohydrates, amino acids, and fatty acids.

The chemoenzymatic synthesis of new surfactants is reported; they were prepared from unprotected carbohydrates, amino acids, and fatty acids. This study pointed out the factors that govern the possibility to enzymatically bind the carbohydrate to the amino acid.

Two key chiral intermediates in a new 4-hydroxyisoleucine synthesis.

We present the crystal and molecular structure of two key compounds of a new synthesis strategy for isomers of natural (2S,3R, 4S)-4-hydroxyisoleucines, 2,3,5,6,7, 8-hexahydro-3-(1-hydroxy-1-methyl-2-oxopropyl)-6,8-methano-7,7, 8a-trimethyl-5H-1,4-benzoxazin-2-one, C(16)H(23)NO(4), and 2,3,5,6,7, 8-hexahydro-3-(1-methyl-2-oxopropyl)-6,8-methano-7,7, 8a-trimethyl-5H-1,4-benzoxazin-2-one, C(16)H(23)NO(3). A new optically pure chiral oxazinone auxiliary derived from (1R,2R, 5R)-2-hydroxypinan-3-one was used.

Postprandial metabolism and aversive response in rats fed a threonine-devoid diet.

Lack of an indispensable amino acid in the diet induces a rapid reduction in food intake. In this study, we assessed whether the anorectic signal after ingestion of a meal lacking threonine originated from either direct perception of the decrease in plasma threonine or from an indirect effect related to increased postprandial amino acid catabolism and energy expenditure. We observed that 3 g of such a meal was sufficient to induce an aversive response to the diet within 2 h. Postprandial changes to plasma ammonia and urea, urinary urea, and energy metabolism did not differ from those measured after a control meal. In contrast, plasma threonine levels fell within 1 h after the meal. It is concluded that an increase in postprandial energy expenditure is not involved in the anorectic response to eating a threonine-devoid diet. The drop in plasma threonine levels may be a potential signal, but the fact that the decrease in food intake occurred 1 h after the decrease in plasma threonine questions a direct causal relationship.

Diastereoselective synthesis of non-proteinogenic alpha-amino acids.

The synthesis of enantiomerically pure azatyrosine, tribromophenylalanine and trichlorophenylalanine is described, using two methods, diastereoselective alkylation and (or) diastereoselective protonation of chiral enolates.

Some of the amino acid chemistry going on in the Laboratory of Amino Acids, Peptides and Proteins.

Some of the chemistry of amino acids going on in our laboratory (Laboratoire des Amino acides Peptides et Protéines) is described as well as some mass spectrometry methodology for their characterization particularly on solid supports. Several aspects are presented including: (i) the stereoselective synthesis of natural and unnatural amino acids using 2-hydroxypinan-3-one as chiral auxiliary; (ii) the stereoselective synthesis of natural and unnatural amino acids by deracemization of alpha-amino acids via their ketene derivatives; (iii) the synthesis of alpha-aryl-alpha-amino acids via reaction of organometallics with a glycine cation; (iv) the diastereoselective synthesis of glycosyl-alpha-amino acids; (v) the synthesis of beta-amino acids using alpha-aminopyrrolidinopiperazinediones as chiral templates; (vi) the reactivity of urethane-N-protected N-carboxyanhydrides. To characterize natural and non natural amino acids through their immonium ions by mass spectrometry, some methodology is also described.

Satiety related to 24 h diet-induced thermogenesis during high protein/carbohydrate vs high fat diets measured in a respiration chamber.

OBJECTIVE: Assessment of a possible relationship between perception of satiety and diet-induced thermogenesis, with different macronutrient compositions, in a controlled situation over 24 h. DESIGN: Two diets with different macronutrient compositions were offered to all subjects in randomized order. SETTING: The study was executed in the respiration chambers at the department of Human Biology, Maastricht University. SUBJECTS: Subjects were eight females, ages 23-33 y, BMI 23+/-3 kg/m2, recruited from University staff and students. INTERVENTIONS: Subjects were fed in energy balance, with protein/carbohydrate/fat: 29/61/10 and 9/30/61 percentage of energy, with fixed meal sizes and meal intervals, and a fixed activity protocol, during 36 h experiments in a respiration chamber. The appetite profile was assessed by questionnaires during the day and during meals. Diet induced thermogenesis was determined as part of the energy expenditure. RESULTS: Energy balance was almost complete, with non-significant deviations. Diet-Induced-Thermogenesis (DIT) was 14.6+/-2.9%, on the high protein/carbohydrate diet, and 10.5+/-3.8% on the high fat diet (P < 0.01). With the high protein/high carbohydrate diet, satiety was higher during meals (P < 0.001; P < 0.05), as well as over 24 h (P < 0.001), than with the high fat diet. Within one diet, 24 h DIT and satiety were correlated (r = 0.6; P < 0.05). The difference in DIT between the diets correlated with the differences in satiety (r = 0.8; P < 0.01). CONCLUSION: In lean women, satiety and DIT were synchronously higher with a high protein/high carbohydrate diet than with a high fat diet. Differences (due to the different macronutrient compositions) in DIT correlated with differences in satiety over 24 h.

Diet induced thermogenesis measured over 24h in a respiration chamber: effect of diet composition.

OBJECTIVE: To study the effect of diet composition on diet-induced thermogenesis (DIT) over 24h in a respiration chamber. SUBJECTS: Eight healthy female volunteers (age 27 +/- 3 y; body mass index, BMI 23 +/- 3 kg/m2). DIETS: A high protein and carbohydrate (HP/C) (60:10:30; percentage energy (E%)carbohydrate, fat and protein, respectively) and high fat (HF) (30:60:10 respectively) diet, both isoenergetic, isovolumetric, composed of normal food items and matched for organoleptic properties (taste, smell, appearance). DESIGN: Subjects spent two 36h periods each in a respiration chamber consuming both test diets in random order. Components of 24h energy expenditure (24h EE): sleeping metabolic rate, DIT and activity induced energy expenditure were measured. RESULTS: DIT was higher in all subjects while on the HP/C diet (1295 kJ/d vs 931 kJ/d; 14.6% vs 10.5% of energy intake; P < 0.02). There was no significant difference in other components or total 24h EE, although there was a trend towards higher EE on the HP/C diet. CONCLUSION: A high protein and carbohydrate diet induces a greater thermic response in healthy individuals when compared to a high fat diet.

Production of polyclonal antibodies towards the immunodetection of insecticide phosalone.

Hapten synthesis for the production of specific insecticide phosalone polyclonal antibodies was carried out starting from an intermediate of the phosalone synthesis, 6-chloro-2-benzoxazolone 1. Two haptens containing different spacers have been prepared: N-5-carboxypentyl-6-chloro-2-benzoxazolone 7 and N-(2-oxo-3-aza-5-carboxypentyl)-6-chloro-2-benzoxazolone 12. Each of these two haptens conjugated to bovine serum albumine (BSA) was used to immunize four rabbits. Immunoassays of phosalone were performed with ELISA using solid-phase bound hapten thyroglobulin conjugate and horseradish peroxidase labelled goat antirabbit IgG. The more sensitive response was observed when the antiserum obtained from the rabbit immunized with the hapten-BSA conjugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competition with the same hapten coupled to thyroglobulin. An identical response was obtained under the same conditions when using benzoxazolone instead of phosalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating conjugate concentration (from 2 to 0.05 micrograms/well) and also the use of heterologous coating protein instead of homologous did improve the sensitivity, resulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.

Leptin receptor gene in a large cohort of massively obese subjects: no indication of the fa/fa rat mutation. Detection of an intronic variant with no association with obesity.

The massive obesity caused in rodents by the disruption of the leptin-receptor signal through genetic defects at the level of either leptin (OB) or leptin receptor (OB-R) has raised the question of the relevance of these genes to morbid obesity in humans. In this study, we screened a large population of massively obese subjects for the presence of a leptin receptor mutation homologous to that of fa/fa rats, a single base substitution changing glutamine 269, a highly conserved glutamine found at position 270 in the human sequence. After polymerase chain reaction (PCR) amplification of a DNA region encompassing the end of exon 5, intron 5, and the beginning of exon 6, we performed restriction fragment length polymorphism analysis. Within the limitations of this approach where only mutations introducing restriction sites (5 of 8 possibilities) could be assessed, no evidence of mutation at the codon gln 270 was found in 343 massively obese subjects. However, a new OB-R gene variant in intron 5 was revealed by MaeII digestion of the PCR products. MaeII/hOB-R genotyping revealed no difference in the distribution of the genotypes between obese subjects and a group of 79 unrelated nonobese control subjects. In addition, no significant association between various obesity-related metabolic phenotypes and the presence of MaeII/hOB-R alleles was found. Thus, our results did not support a significant role for the MaeII/hOB-R gene variant in the development of the obese phenotype in the population we studied.

A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes. Implication In the overactivity of FAS promoter in obese Zucker rats.

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from -200 to -126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells. This region, fused to a heterologous promoter, the E1b TATA box, induced differential levels of gene reporter activity in lean and obese rat adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat genotype, adipocyte nuclear proteins were shown to footprint the same protected sequence within the fa-responsive region, and supershift analysis demonstrated that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling evidence that the Sp1 binding site contained in this sequence was implicated in the differential promoter activity in lean versus obese rats, was provided by the observation that mutations at this Sp1 site induced a 2.5-fold increase in FAS promoter activity in adipocytes from lean rats, whereas they had no effect in adipocytes from obese rats.

[Contribution of post mortem skull scanning for the study of sudden infant death]. / Contribution du scanner crânien post-mortem à l'étude de la mort subite du nourrisson.

BACKGROUND: Despite post-mortem examination and autopsy, many cases of sudden infant death (SID) remain unexplained. The aim of this study was to assess usefulness of CT-scan in the Sudden Infant Death Syndrome (SIDS). POPULATION: Twenty-three cases of SIDS had a post-mortem CT-scan evaluation of skull and brain. The pictures were retrospectively reviewed by several independent radiologists who were unaware of the circumstances of death and results of autopsy. RESULTS: Aspects of pneumatocele, probably due to lumbar puncture were found in 6 cases. The subarachnoid spaces appeared inexplically hyperdense, as they were not correlated to the results of lumbar puncture and autopsy. The ventricles were normal in size or density. Density of the dural sinuses (superfical and deep) was often increased, an aspect possibly artefactual, due to post-mortem thrombosis. The cerebral parenchyma was often slighty hypodense; microcalcifications due to congenital toxoplasmosis were found in one case. CONCLUSION: There was no correlation between the CT-scan imaging and the delay of death and lumbar puncture. Infants with or without subarachnoid hemorrage had the same CT scan findings. The CT-scan has a poor value when autopsy is performed; in its absence, it could be useful for diagnosing post-traumatic intracerebral hematoma.

C/EBP alpha expression in adipose tissue of genetically obese Zucker rats.

The adipose tissue of genetically obese Zucker rats is characterized by coordinated tissue specific overtranscription of a subset of genes related to lipid storage such as Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). We show that CCAAT/Enhancer Binding Protein alpha (C/EBP alpha) is an activator of GAPDH proximal promoter in transiently transfected mature rat adipocytes. C/EBP alpha mRNA levels were increased in adipose tissue but not in liver of obese as compared to lean rats at 30 days of age, i.e., when obesity is fully expressed. Nevertheless at 16 days of age, although overdevelopment of adipose tissue could be detected in preobese rats, C/EBP alpha mRNA levels were similar whatever the genotype. In conclusion C/EBP alpha mRNA is overexpressed in adipose tissue of obese rats, suggesting a possible role for this factor in the activation of lipid storage-related genes in adipose tissue of obese rats. However, C/EBP alpha overexpression is not temporally related to the onset of obesity.

Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats.

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.

[Comparative contribution of endoscopy, x-ray computed tomography and MRI in the preoperative evaluation of cancers of the endolarynx: apropos of 18 surgically-treated patients]. / Apports comparés de l'endoscopie, de la tomodensitométrie et de l'I.R.M. dans le bilan pré-opératoire des cancers de l'endolarynx: à propos de 18 patients opérés.

The authors carried out a prospective and comparative study of 18 patients with endolaryngeal carcinoma, with preoperative imaging of the extent of the disease by computed tomography (CT) and magnetic resonance imaging (MRI). The correlations between CT, MRI, endoscopy and postoperative histology are reported. It appears to the authors that the contribution of imaging is evident and that at present the reliability of CT is comparable to that of MRI, taking account of the current technical problems of MRI.