Strategies to identify transport systems in plants.

Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation. The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems. A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.

Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins.

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.

An analytical model accounting for tip shape evolution during atom probe analysis of heterogeneous materials.

An analytical model describing the field evaporation dynamics of a tip made of a thin layer deposited on a substrate is presented in this paper. The difference in evaporation field between the materials is taken into account in this approach in which the tip shape is modeled at a mesoscopic scale. It was found that the non-existence of sharp edge on the surface is a sufficient condition to derive the morphological evolution during successive evaporation of the layers. This modeling gives an instantaneous and smooth analytical representation of the surface that shows good agreement with finite difference simulations results, and a specific regime of evaporation was highlighted when the substrate is a low evaporation field phase. In addition, the model makes it possible to calculate theoretically the tip analyzed volume, potentially opening up new horizons for atom probe tomographic reconstruction.

Analogies between optical propagation and heat diffusion: applications to microcavities, gratings and cloaks.

A new analogy between optical propagation and heat diffusion in heterogeneous anisotropic media has been proposed recently by three of the present authors. A detailed derivation of this unconventional correspondence is presented and developed. In time harmonic regime, all thermal parameters are related to optical ones in artificial metallic media, thus making possible to use numerical codes developed for optics. Then, the optical admittance formalism is extended to heat conduction in multilayered structures. The concepts of planar microcavities, diffraction gratings and planar transformation optics for heat conduction are addressed. Results and limitations of the analogy are emphasized.

Do plastid envelope membranes play a role in the expression of the plastid genome?

A unique biochemical machinery is present within the two envelope membranes surrounding plastids (Joyard et al., Plant Physiol. 118 (1998) 715-723) that reflects the stage of development of the plastid and the specific metabolic requirements of the various tissues. Envelope membranes are the site for the synthesis and metabolism of specific lipids. They are also the site of transport of metabolites, proteins and information between plastids and surrounding cellular compartments. For instance, a complex machinery for the import of nuclear-encoded plastid proteins is rapidly being elucidated. The functional studies of plastid envelope membranes result in the characterization of an increasing number of envelope proteins with unexpected functions. For instance, recent experiments have demonstrated that envelope membranes bind specifically to plastid genetic systems, the nucleoids surrounded by plastid ribosomes. At early stages of plastid differentiation, the inner envelope membrane contains a unique protein (named PEND protein) that binds specifically to plastid DNA. This tight connection suggests that the PEND protein is at least involved in partitioning the plastid DNA to daughter plastids during division. The PEND protein can also provide a physical support for replication and transcription. In addition, factors involved in the control of plastid protein synthesis can become associated to envelope membranes. This was shown for a protein homologous to the E. coli ribosome recycling factor and for the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins. In fact, the envelope membranes together with the plastid DNA are the two essential constituents of plastids that confer identity to plastids and their interactions are becoming uncovered through molecular as well as cytological studies. In this review, we will focus on these recent observations (which are consistent with the endosymbiotic origin of plastids) and we discuss possible roles for the plastid envelope in the expression of plastid genome.

[Cost of early management of chronic inflammatory intestinal disease]. / Coût de la première prise en charge des maladies inflammatoires chroniques de l'intestin.

OBJECTIVES: The aim of this study was to assess the cost of the first management of inflammatory bowel disease (IBD) from the onset of first symptoms until 6 weeks after the diagnosis. This cost was calculated in French francs (FF) for all IBD and namely for Crohn's disease (CD), ulcerative colitis (UC), and ulcerative proctitis (UP). MATERIAL AND METHODS: Data concerning 258 patients were collected by the mean of a standardized questionnaire from 3 different sources: the patient, his general practitioner, and his gastroenterologist. RESULTS: Two hundred and fifty eight patients were included: 144 CD (55.8%), 76 UC (29.5%), 30 UP (11.6%), and 8 chronic unclassifiable colitis (CUC) (3.1%). The mean direct costs of the diagnosis (m +/- SD) were 23,116 +/- 40,820 FF for CD, 10,628 +/- 17,316 FF for UC and 3,451 +/- 2,743 FF for UP. Although unplanned hospitalizations occurred in only 38% of the patients (98/258), they represented the 3/4 of the mean costs: 78.2% for CD and 64% for UC. Indirect costs generated by days off work were 4,719 +/- 6,610 FF for CD, 2,996 +/- 6,897 FF for UC and 1,230 +/- 3,622 FF for UP. CONCLUSION: The first management of a patient with CD was twice more expensive than the one with UC and 6.5 times than the one with UP.

[Consequences of obliterating arteriopathy of the legs on the professional activity of patients and external assistance]. / Conséquences de l'artériopathie oblitérante des membres inférieurs (AOMI) sur l'activité professionnelle des patients et sur les aides extérieures.

Peripheral arterial occlusive disease of the lower limbs results in increased risks of cardiovascular and cerebrovascular morbidity and mortality. However, peripheral arterial disease also has an incapacitating effect upon patients' daily lives, and indeed can have considerable consequences upon their quality of life as well as upon their professional activities. The purpose of this study was to describe patients in terms of their professional activities, the consequences of the disease upon such activities, need for external assistance, and the effects of treatment over a six-month treatment follow-up period. This was a prospective, randomised, double-blind, multicentre study comparing naftidrofuryl (three 200-mg tablets per day) versus placebo in patients presenting with stage-II peripheral arterial disease. Four study visits were scheduled for each patient: an initial selection visit, followed by a 30-day placebo wash-out period, an inclusion visit (to allow verification of inclusion and non-inclusion criteria), and follow-up visits after 3 and 6 months of treatment. 234 patients were included in the study in intention to treat (117 patients in the naftidrofuryl group and 117 patients in the placebo group). These two groups were entirely comparable for all criteria (sociodemographic criteria, risk factors, cardiovascular history, duration of disease) except for obesity and dyslipidaemia, for which prevalence was higher in the naftidrofuryl group. 12.4% of the study population were professionally active at the time of the survey (mean age: 65.9 years). However, 42.3% of patients had in fact been active at the time when arterial disease became manifest. Of these patients, 45.4% (45 patients) stated that arterial disease had affected their professional activity: 28.9% (13 patients) reported a change in their activity, 26.7% (12 patients) reported partial suspension of their activity, and 44.4% (20 patients) stated that all professional activities had ceased. Changes in professional activity comprised invalidity (36.2%), prolonged sick leave (25.5%), premature retirement (14.9%), sick leave (17%), unemployment (6.4%), reduction in long-distance travel (4.3%), change of job (8.5%) or salary reduction (2.1%). During the 6-month follow-up period, only 4 patients were on sick leave (3 in the placebo group and 1 in the naftidrofuryl group). Furthermore, less than 10% of the study population required external assistance as a result of the disease. This analysis thus highlights the repercussions of this disease upon the patients' daily and professional activities.

[Profiles of 939 patients with obstructive sleep apnea syndrome treated with continuous positive pressure]. / Typologie de 939 patients traités par pression positive continue pour syndrome d'apnées obstructives du sommeil.

The aim of this study was to analyze the characteristics of patients on long-term continuous positive pressure therapy for obstructive sleep apnea in order to determine the efficacy, observance, tolerance, degree of patient satisfaction and patient quality of life using the Nottingham scale. A questionnaire was sent to 939 patients treated for at least six months in 11 centers. Factorial analysis of multiple correspondences and two classification analyses were used to establish patient profiles. Factorial analysis evidenced a relationship between patient satisfaction, reduced symptoms, observance and tolerance. The classification analyses distinguished three groups. Group A (n = 596) included primarily men (93%) who were satisfied with the treatment (99%) and showed good observance. Group B (n = 284) was characterized by patient satisfaction, observance, improved symptoms and lower quality of life than group A. Group C was composed of older patients who were satisfied with their treatment and showed good observance but who had no notable improvement in their symptoms. In conclusion, this study pointed out the difficulty in defining which patients with obstructive sleep apnea would benefit most from continuous positive pressure therapy. Good observance is not a sufficient criterion for therapeutic efficacy.

A solid-mounted resonator-oscillator-based 4.596 GHz frequency synthesis.

This paper describes a 4.596 GHz frequency synthesis based on a 2.1 GHz solid mounted resonator (SMR) voltage-controlled oscillator (VCO). The SMR oscillator presents a chip size lower than 2 mm(2), a power consumption of 18.2 mW, and exhibits a phase noise of -89 dBc/Hz and -131 dBc/Hz at 2 kHz and 100 kHz offset frequencies, respectively. The VCO temperature-frequency dependence is measured to be -14 ppm∕°C over a range of -20°C to 60°C. From this source, a low noise frequency synthesizer is developed to generate a 4.596 GHz signal (half of the Cs atom hyperfine transition frequency) with a phase noise of -81 dBc/Hz and -120 dBc/Hz at 2 kHz and 100 kHz from the carrier. The frequency synthesis output is used as a local oscillator in a Cs vapor microcell-based compact atomic clock. Preliminary results are reported and discussed. To the authors knowledge, this is the first development of a SMR-oscillator-based frequency synthesizer for miniature atomic clocks applications.

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence.

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.

Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?

A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme.

Do Ringtailed lemurs (Lemur catta) reconcile in the hour post-conflict?: A pilot study.

A previous study byKAPPELER (1993) showed that no reconciliation occurred in the ringtailed lemur (Lemur catta). Like the majority of studies dealing with reconciliation, the post-conflict period lasted 10 minutes. An identical study using a 70-min post-conflict period was conducted on one of the same semi-free ranging group studied six years earlier byKAPPELER. Results showed that individuals who never interacted within 10 min did engage in friendly interactions in the longer lasting postconflict period.

O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform.

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.

A new chloroplast envelope carbonic anhydrase activity is induced during acclimation to low inorganic carbon concentrations in Chlamydomonas reinhardtii.

Using mass-spectrometric measurements of 18O exchange from 13C18O2 we determined the activity of carbonic anhydrase (CA; EC 4.2.1.1) in chloroplast envelope membranes isolated from Chlamydomonas reinhardtii cw-15. Our results show an enrichment of CA activity in these fractions relative to the activity in the crude chloroplast. The envelope CA activity increased about 8-fold during the acclimation to low-CO2 conditions and was completely induced within the first 4 h after the transfer to air levels of CO2. The CA-activity was not dissociated from envelope membranes after salt treatment. In addition, no cross-reactivity with other CA isoenzymes of Chlamydomonas was observed in our chloroplast envelope membranes. All these observations indicated that the protein responsible for this activity was a new CA isoenzyme, which was an integral component of the chloroplast envelopes from Chlamydomonas. The catalytic properties of the envelope CA activity were completely different from those of the thylakoid isoenzyme, showing a high requirement for Mg2+ and a high sensitivity to ethoxyzolamide. Analysis of the integral envelope proteins showed that there were no detectable differences between high- and low-inorganic carbon (Ci) cells, suggesting that the new CA activity was constitutively expressed in both high- and low-Ci cells. Two different high-Ci-requiring mutants of C. reinhardtii, cia-3 and pmp-1, had a reduced envelope CA activity. We propose that this activity could play a role in the uptake of inorganic carbon at the chloroplast envelope membranes.

Sulfolipid is a potential candidate for annexin binding to the outer surface of chloroplast.

Using a subcellular-specific proteomic approach, we have identified by protein microsequencing, a putative 35-kDa annexin from among the chloroplast envelope polypeptides. To confirm this identification, we demonstrate that (a) a 35-kDa protein, identified as annexin by antibody cross-reactivity, co-purifies with Percoll-purified chloroplasts and their envelope membranes when extracted in the presence of Ca(2+) and (b) the native spinach annexin protein binds to chloroplast-specific lipids in a Ca(2+)-dependent manner. The binding of the spinach annexin to these glycerolipids occurs at similar Ca(2+) concentrations as those, which promote the interaction of annexins to phospholipids in other membranes. Among chloroplast glycerolipids known to be accessible on the cytosolic face (outer leaflet) of the outer envelope membrane, sulfolipid, and probably phosphatidylinositol, would be the sole candidates for a putative Ca(2+)-dependent interaction of annexin with the chloroplast surface.

Spinach chloroplast 0-acetylserine (thiol)-lyase exhibits two catalytically non-equivalent pyridoxal-5'-phosphate-containing active sites.

A synthetic gene encoding the mature spinach- chloroplast O-acetylserine (thiol)-lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co-substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoacrylate and the pyridoxal 5'-phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with the acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the enzyme exhibits two non-equivalent pyridoxal-5'-phosphate-containing active sites.

Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins.

As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid-interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

Epidemiological survey of the orthopaedic status of severe haemophilia A and B patients in France. The French Study Group. secretariat.haemophiles@cch.ap-hop-paris.fr.

One hundred and 16 patients contributed to an analysis of the impact of the consequences of severe haemophilia A or B (factor levels < 2%) on orthopaedic status, resources consumed in relation to this status and resultant cost, and quality of life as perceived by the patient, using the MOS 36-Item-Short-Form Health Survey (SF-36). This French cross-sectional study involved outpatients regularly attending a haemophilia treatment centre. Data were collected retrospectively over a period of 1 year by the physician of the haemophilia treatment centre. Patients had a mean age of 23, and consisted of 50% students, 25% salaried workers, 17.2% with no professional activity and 7.8% physically impaired; 82.8% of them had type A haemophilia. Mean pain score was 2.5 per patient for the six main joints; 7.7 for the clinical score and 18.8 for the radiological score, with a mean number of bleeds of 16.3 per year per patient. During the year prior to inclusion, and because of their orthopaedic status, 22.4% of patients were hospitalized, 76.7% attended for an outpatient visit and 76.7% required at least one special investigation; 97.4% received replacement therapy, 41.4% required treatment for joint pain and 42.2% orthopaedic equipment. The less affected dimensions were the physical function (76.8 +/- 22. 2) and the social relations (76.1 +/- 23.1). Least good quality of life scores concerned the pain (60.2 +/- 25.2), perception of general health (59.3 +/- 23.1) and vitality (57.8 +/- 19.5) dimensions. The age was a discriminant criterion since quality of life was better in patients of the 18-23 age group for five dimensions. Mean annual treatment costs of a patient with severe haemophilia were determined as 425 762 French francs ($73 029). Loss of production was estimated at a mean of 4609 French francs ($791) per active patient over the course of the year. Results showed indirect evidence of the usefulness of early home treatment.