An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors.

KEY MESSAGE: A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay. The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora ß-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

Exploring ITM2A as a new potential target for brain delivery.

BACKGROUND: Integral membrane protein 2A (ITM2A) is a transmembrane protein expressed in a variety of tissues; little is known about its function, particularly in the brain. ITM2A was found to be highly enriched in human brain versus peripheral endothelial cells by transcriptomic and proteomic studies conducted within the European Collaboration on the Optimization of Macromolecular Pharmaceutical (COMPACT) Innovative Medicines Initiative (IMI) consortium. Here, we report the work that was undertaken to determine whether ITM2A could represent a potential target for delivering drugs to the brain. METHODS: A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization studies in Human Embryonic Kidney 293 (HEK293) cells overexpressing ITM2A and in brain microvascular endothelial cells from mouse and non-human primate (NHP) were performed with these tools. The best ITM2A antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in an in vivo mouse pharmacokinetic study to investigate its ability to cross the BBB. RESULTS: Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-overexpressing cells. However, despite high RNA expression in mouse and human microvessels, the ITM2A protein was rapidly downregulated when endothelial cells were grown in culture, probably explaining why transcytosis could not be observed in vitro. An attempt to directly demonstrate in vivo transcytosis in mice was inconclusive, using either a cross-reactive anti-ITM2A antibody or in vivo phage panning of an anti-ITM2A phage library. CONCLUSIONS: The present work describes our efforts to explore the potential of ITM2A as a target mediating transcytosis through the BBB, and highlights the multiple challenges linked to the identification of new brain delivery targets. Our data provide evidence that antibodies against ITM2A are internalized in ITM2A-overexpressing HEK293 cells, and that ITM2A is expressed in brain microvessels, but further investigations will be needed to demonstrate that ITM2A is a potential target for brain delivery.

[Evaluation of the clinical and radiological results of cervical longitudinal median somatotomy without graft]. / Evaluation des résultats cliniques et morphologiques de la somatotomie cervicale médiane longitudinale sans greffe.

OBJECTIVES: To evaluate the clinical and radiological results of cervical longitudinal median somatotomy without graft, used for the treatment of cervical myelopathy and radiculopathy, and compare it to techniques with graft and to laminectomies. MATERIAL AND METHOD: Thirty-four patients (25 males and nine females), with a mean age over 60 years, were included in a study comparing pre- and postoperative clinical status on the Japanese Orthopaedic Association (JOA) functional scale and radiological status with evaluation of the cervical curve on plain films and dynamic tests in flexion and extension. RESULTS: No significant difference was found with the clinical and anatomical results published in the literature concerning median somatotomies performed with graft and/or osteosynthesis and laminectomies and their variants. CONCLUSIONS: The cervical longitudinal median somatotomy without graft is an easy and reliable technique that can be proposed as first-line treatment for cervical spondylotic myelopathy related to anterior compression. It decreases the cost and the duration of the surgical procedure, it protects the patient from the complications and sequelae related to graft harvesting and the use of implants. It should be limited to patients without preoperative kyphosis who are over 50 years old.

Deciphering the immune microenvironment of a tissue by digital imaging and cognition network.

Evidence has highlighted the importance of immune cells in various gut disorders. Both the quantification and localization of these cells are essential to the understanding of the complex mechanisms implicated in these pathologies. Even if quantification can be assessed (e.g., by flow cytometry), simultaneous cell localization and quantification of whole tissues remains technically challenging. Here, we describe the use of a computer learning-based algorithm created in the Tissue Studio interface that allows for a semi-automated, robust and rapid quantitative analysis of immunofluorescence staining on whole colon sections according to their distribution in different tissue areas. Indeed, this algorithm was validated to characterize gut immune microenvironment. Its application to the preclinical colon cancer APCMin/+ mouse model is illustrated by the simultaneous counting of total leucocytes and T cell subpopulations, in the colonic mucosa, lymphoid follicles and tumors. Moreover, we quantify T cells in lymphoid follicles for which quantification is not possible with classical methods. Thus, this algorithm is a new and robust preclinical research tool, for investigating immune contexture exemplified by T cells but it is also applicable to other immune cells such as other myeloid and lymphoid populations or other cellular phenomenon along mouse gut.

Construction and characterization of a catalytic fusion protein system: P-450(11beta)-adrenodoxin reductase-adrenodoxin.

Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11beta-hydroxylation of 11-deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11beta). Various fusion constructs of P-450(11beta) with its electron transfer components, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both adrenodoxin reductase and adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11beta) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11beta)-adrenodoxin reductase-adrenodoxin was more active than that of P-450(11beta)-adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P-450(11beta), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11beta)-adrenodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production.

Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide.

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings.

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.

A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb.

We have isolated complementary DNA clones encoding a novel orphan member of the nuclear receptor superfamily, termed BD73. This protein shows strong amino acid sequence similarity to the previously described Rev-ErbA alpha. Unlike Rev-Erb, in which the opposite strand of the C-terminal coding region encodes the C-terminal portion of a variant thyroid hormone receptor isoform, the opposite strand of the C-terminal coding region of BD73 does not have any extensive open reading frames. BD73 messenger RNA is expressed in a wide variety of tissues and cell lines. In quiescent HepG2 cells, BD73 messenger RNA levels are strongly induced by planar aromatic antioxidants. Like Rev-Erb, BD73 binds as a monomer to a DNA sequence which consists of a specific A/T-rich sequence upstream of the consensus hexameric half-site specified by the P box of the DNA-binding domain. Amino acid sequence comparisons suggest that the A box sequence, which has been suggested to mediate monomer binding by other superfamily members, lies closer to the DNA-binding domain in BD73 and Rev-Erb than in other receptors. Under the conditions examined, neither BD73 nor Rev-Erb activated reporters containing multiple copies of their common binding site. Thus, these two orphans may require an as yet unidentified ligand or other signal for such activation. Together, BD73 and Rev-Erb define a subgroup of orphan receptors that bind as monomers to a half-site flanked by a specific and extended A/T-rich sequence.

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.

A gene encoding a yeast equivalent of mammalian NADPH-adrenodoxin oxidoreductases.

Adrenodoxin oxidoreductase (ADR) and adrenodoxin (ADX) are the two proteins involved in electron transport to mammalian mitochondrial P-450s capable of steroid modifications. The cloning and sequencing of a S. cervisiae ADR homologue (YADR) is presented here. The YADR protein sequence shares 36 and 37% of identical amino acids with human and bovine ADR respectively. The physiological role of this ADR homologue in yeast is unknown. We intend to study the interaction of this YADR with bovine ADX in vitro and in vivo.

Isolation and sequence analysis of Clpg1, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum.

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race beta and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed on ORF encoding a 363-amino-acid (aa) polypeptide beginning with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non-coding region. This genomic clone was thereafter designated Clpg1. Southern analysis, performed with a Clpg1-specific probe, showed that this gene is present as a single copy in the Cl genome.

One-step purification and characterization of a lignin-specific O-methyltransferase from poplar.

O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH). Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P. deltoides). The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose. The enzyme uses both CA and 5OH as substrates. We previously have reported the cloning of a corresponding OMT cDNA [Dumas et al., Plant Physiol. 98 (1992) 796-797]. Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone. Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome. RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves.

Molecular characterization of CLPT1, a SEC4-like Rab/GTPase of the phytopathogenic fungus Colletotrichum lindemuthianum which is regulated by the carbon source.

The gene CLPT1 (Colletotrichum lindemuthianum Protein Transport 1) encoding a Rab/GTPase was isolated from the filamentous fungus Colletotrichum lindemuthianum, the causal agent of bean anthracnose. At the amino acid level, CLPT1 shows between 54 and 80% identity to SEC4-like proteins, a class of molecules required for intracellular vesicular transport in yeasts. In particular, typical SEC4 domains involved in nucleotide binding and membrane attachment are present in the CLPT1 sequence. Functional identity of CLPT1 with SEC4 was confirmed by complementation of the Saccharomyces cerevisiae sec4-8 mutation. This is the first report of a gene involved in the control of intracellular vesicular trafficking in a phytopathogenic fungus. RNA blot analyses of CLPT1 expression were performed during in vitro growth of the fungus on synthetic media containing glucose or pectin, as single carbon source. The accumulation of CLPT1 mRNA was strongly increased on pectin, a plant cell wall polysaccharide that induces the production of extracellular pectinases, whereas the level of CLPT1 mRNA was below the detection threshold on glucose. These results suggest that CLPT1 is mainly involved in protein secretion and that the production of extracellular enzymes potentially involved in pathogenesis in filamentous fungi is sustained by induction of the genes involved in the secretory machinery.

Kinetics of the action of chymosin (rennin) on a peptide bond of bovine alpha s1-casein. Comparison of the behaviour of this substrate with that of beta- and kappa o-caseins.

The action of chymosin on the Phe23-Phe24 bond of bovine alpha s1-casein, in citrate buffer (pH 6.2) at 30 degrees C, was followed by reversed-phase HPLC quantification of residual alpha s1-casein or fragment 24-199 after different time periods and at different substrate concentrations. This allowed determination of the Michaelian parameters for the reaction under study which were compared with those previously obtained for the action of chymosin on beta- and kappa o-casein under identical reaction conditions. The whole efficiency of the three reactions, as estimated by kcat/Km, was 1.8, 20.6 and 1405.0 for alpha s1-, beta- and kappa o-caseins, respectively. The specificity of chymosin is discussed in the light of these results and of the known sequences of the 3 caseins.

[The primary structure of bovine alpha s2 caseins]. / Premiers elements de structure primaire des caseines alphas2 bovines

The bovine alphas2-, alphas3-, alphas4- and alphas6-caseins [1] were isolated. The 4 proteins had the same amino-acid composition and C-terminal sequence, but different phosphorus contents. From a mixture of these proteins (designated as 'alphas2-complex') and from alphas3-casein a single and identical N-terminal sequence was obtained by Edman degradation. It seems therefore that the 4 proteins have the same peptide chain and only differ in their phosphorus content. For this reason we propose to modify the nomenclature of Annan and Mason [1] and to use in future the single term alphas2 to designate the caseins which have been previously called alphas2,alphas3,alphas4 and alphas6 by these authors. The study of the primary structure of the peptide chain, which has confirmed these results, was undertaken on the S-carboxymethylated alphas2-complex. From a cyanogen bromide digest and from a tryptic hydrolyzate of the alphas2-complex, 5 and 25 peptides were obtained respectively, both sets of peptides accounting for the whole peptide chain. Examination of the tryptic peptides containing methionine combined with the N- and C-terminal sequences of the alphas2-complex and some CNBr peptides, gave the order of the CNBr peptides, H.CN4--CN2--CN5--CN1--CN3.OH, which contain 4, 22, 115, 49 and 17 residues respectively. A partial sequence accounting for half of the peptide chain of the alphas2-complex is given. This peptide chain is likely composed of 207 amino-acid residues.

C-terminal labelling of beta-casein.

This paper is the first to report specific labelling of a native protein at its C-terminal end by carboxypeptidase Y-catalyzed transpeptidation between beta-casein and tritiated Phe amide. A tryptic digest of the radiolabelled protein was resolved by reversed-phase HPLC and a single labelled peptide was isolated therefrom. Sequence determination and FAB mass spectrometry showed that the last 2 residues (Val-209, Ile-208) of beta-casein had been deleted and Ile 207 substituted by Phe, deamidation presumably occurring after transpeptidation. Identical results were obtained by transpeptidating the isolated C-terminal tryptic heptapeptide (203-209) of native beta-casein.

A new strategy for primary structure determination of proteins: application to bovine beta-casein.

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C-terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS-PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N-terminal sequence of peptide (i-1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine beta-casein (209 residues) which was completely resequenced with only 239 Edman cycles.

Preparation and amino acid sequence of human kappa-casein.

Human kappa-casein was prepared from whole casein by successive hydroxyapatite and thiol-Sepharose chromatographies. The primary structure of its 99-residue N-terminal fragment has been determined by sequencing peptides obtained by tryptic and chymotryptic digestions of the whole protein. This fragment overlaps the known sequence of the 65-residue C-terminal fragment. The 158-residue sequence of human kappa-casein was compared to those of goat, ewe, cow and rat kappa-caseins. Only 22% of the residues are identical in homologous positions. The rate of divergence of the 93-residue N-terminal segment (para-kappa-casein) appears to be higher than that of the rest of the molecule.

Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

Study of the heterogeneity of bovine pepsin A by pseudo isoelectric focusing.

Pseudo isoelectric focusing (PIEF), i.e. focusing stopped before the equilibrium is reached, has been successfully applied to analyse bovine pepsins A obtained from abomasum mucosa extracts and from 3 individual abomasal juices. Whole bovine pepsin A from mucosa extracts, was resolved in PIEF, using an acidic pH gradient(2-4), into five major and active components. Its fractionation on hydroxyapatite only gave four components which were not homogeneous in PIEF, suggesting that interactions between the different components occur during the chromatographic procedure. After treatment with potato acid phosphatase, whole bovine pepsin A showed only one band in PIEF, displaying enzymic activity and with a mobility identical to that of the less anodic band in untreated pepsin. These results, together with organic phosphate determinations, obviously confirm that the heterogeneity of bovine pepsin A is due to the phosphate content which appears to range between 0 and 3 mol./mol. for the 5 components. Two of them, the phosphate content of which appears to be 1 mol./mol., seem to differ in the location of this phosphate group in the molecule. Whole bovine pepsin A preparations from abomasal juices shared the same pattern in PIEF, identical to that observed with bovine pepsin A from mucosa extracts, thus excluding that dephosphorylation might be involved in the secretory process, as well as disproving the theory that dephosphopepsinogen might be an intermediate in the synthesis of pepsinogen, as it was previously suggested.