Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Osteodentine and vascular osteodentine of Anarhichas lupus (L.)

Summary TEM, SEM and X-ray diffraction analysis demonstrate the heterogeneity of the dentinal tissue of Anarhichas lupus, a vascular osteodentine. The disordered aspect of collagen fibres, incompletely mineralized (the periodical striation being still visible), explains the scattered distribution of crystallites since they are responsible for their arrangement. The low degree of mineralization revealed by the visible collagen striation is confirmed by X-ray diffraction analysis (the crystallinity of vascular osteodentine being much lower than that of the peripheral dental tissue) as well as by high resolution TEM, since no lattice planes could be observed. Osteodentine, supporting bone and proper bone have in common a mineral phase, more or less organized, different from the apatite system.The authors thank Mireille Cottrel-Gengoux for her technical assistance     

Identification of rat neurophysins: Complete amino acid sequences of MSEL- and VLDV-neurophysins

Two rat neurophysins have been purified by salt precipitation, molecular sieving and ion-exchange chromatography. The proteins, performic-acid oxidized or reduced-alkylated, have been split either by trypsin or by staphylococcal proteinase and fragments have been separated by peptide mapping. Amino acid sequences of tryptic peptides have been determined either directly or after cleaving the large fragments by subtilisin, chymotrypsin, elastase or staphylococcal proteinase and characterizing the subfragments. Tryptic peptides have been ordered through the fragments given by staphylococcal proteinase. The N-terminal sequences of both proteins have also been established by automated degradation.The two usual types of mammalian neurophysins have been identified. One neurophysin belongs to the MSEL-neurophysin family and shows 11 substitutions and a 2-residue C-terminal truncation when compared with bovine MSEL-neurophysin. The other belongs to the VLDV-neurophysin family and shows 8 substitutions when compared with bovine VLDV-neurophysin. There are 23 differences between the MSEL- and VLDV-neurophysins of the rat.     

Impact of hyperthermic intraperitoneal chemotherapy on Hsp27 protein expression in serum of patients with peritoneal carcinomatosis

Despite the strong rationale for combining cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal carcinomatosis, thermotolerance and chemoresistance might result from heat shock protein overexpression. The aim of the present study was thus to determine whether the heat shock protein 27 (Hsp27), a potential factor in resistance to treatment, could have a higher level in serum from patients under this combined therapy. Patients receiving CRS plus HIPEC for peritoneal carcinomatosis (group 1), patients with cancer or a history of cancer undergoing abdominal surgery (group 2), and patients without malignancies undergoing abdominal surgery (group 3) were included. Hsp27 serum levels were determined before and at different times following CRS and HIPEC using enzyme-linked immunosorbent assay. In group 1 (n = 25), the high Hsp27 levels, observed at the end of surgery compared with before (p < 0.0001), decreased during HIPEC, but remained significantly higher than before surgery (p < 0.0005). In groups 2 (n = 11) and 3 (n = 15), surgery did not significantly increase Hsp27 levels. A targeted molecular strategy, inhibiting Hsp27 expression in tumor tissue, could significantly reduce resistance to the combined CRS plus HIPEC treatment. This approach should be further assessed in a clinical phase I trial.     

Flow Cytometric Quantification of All Phases of the Cell Cycle and Apoptosis in a Two-Color Fluorescence Plot

An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.