Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.     

The 2.15 A crystal structure of CG-16, the developmentally regulated homodimeric chicken galectin

Differential developmental regulation of expression, fine-specificity differences in ligand recognition and disparate capacity for homodimerization are characteristics of the two currently known proto-type chicken galectins. The X-ray crystal structure of the first avian galectin, the homodimeric agglutinin from chicken liver (CG-16), has been solved in the absence of ligand in two crystal forms. Although the arrangement of lectin dimers in the two crystals is different, the structure of the monomers and their association into the extended beta-sandwich that characterises the dimer are virtually identical. The fold establishes a beta-sandwich motif composed of a five-stranded and a six-stranded beta-sheet evocative of proto-type mammalian galectins. The carbohydrate-binding site is occupied by six water molecules that take the place of the sugar in the complex. They help to stabilise in the absence of the ligand the spatial arrangement of the amino acid side-chains involved in sugar recognition. Docking of N-acetyllactosamine into the binding site reveals that three of these water molecules, which are in direct contact with the protein, occupy positions equivalent to the key sugar hydroxyl groups, namely the hydroxyls at positions 4 and 6 of the galactose unit and at position 3 of the N-acetylglucosamine unit. Crystallographic data are fully consistent with the binding features in solution previously derived from chemical mapping with deoxy, fluoro and O-methyl derivatives and laser photo-CIDNP (chemically induced dynamic nuclear polarisation) studies. The possible molecular basis for the monomeric character of the chicken intestinal galectin as well as potential mechanisms of oxidative inactivation by disulphide bridging are evaluated on the basis of the given structural information concerning the CG-16 dimer interface and the cysteine residues, respectively.     

Characterization of functional bacterial groups in a hypersaline microbial mat community (Salins-de-Giraud, Camargue, France)

A photosynthetic microbial mat was investigated in a large pond of a Mediterranean saltern (Salins-de-Giraud, Camargue, France) having water salinity from 70 per thousand to 150 per thousand (w/v). Analysis of characteristic biomarkers (e.g., major microbial fatty acids, hydrocarbons, alcohols and alkenones) revealed that cyanobacteria were the major component of the pond, in addition to diatoms and other algae. Functional bacterial groups involved in the sulfur cycle could be correlated to these biomarkers, i.e. sulfate-reducing, sulfur-oxidizing and anoxygenic phototrophic bacteria. In the first 0.5 mm of the mat, a high rate of photosynthesis showed the activity of oxygenic phototrophs in the surface layer. Ten different cyanobacterial populations were detected with confocal laser scanning microscopy: six filamentous species, with Microcoleus chthonoplastes and Halomicronema excentricum as dominant (73% of total counts); and four unicellular types affiliated to Microcystis, Chroococcus, Gloeocapsa, and Synechocystis (27% of total counts). Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments confirmed the presence of Microcoleus, Oscillatoria, and Leptolyngbya strains (Halomicronema was not detected here) and revealed additional presence of Phormidium, Pleurocapsa and Calotrix types. Spectral scalar irradiance measurements did not reveal a particular zonation of cyanobacteria, purple or green bacteria in the first millimeter of the mat. Terminal-restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA gene fragments of bacteria depicted the community composition and a fine-scale depth-distribution of at least five different populations of anoxygenic phototrophs and at least three types of sulfate-reducing bacteria along the microgradients of oxygen and light inside the microbial mat.     

Phylogenetics of Lophodermium from pine

Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.     

Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of normal salivary polypeptides (polypeptides A-B). The appearance of new polypeptides in saliva could be established unequivocally within the 24 h following the trophic adrenergic stimulation. On the other hand, salivary polypeptides induced in response to a single administration of IPR could be demonstrated as late as 7-9 days after the stimulation. Accordingly, detection of parotid polypeptides C-G in PIL-produced saliva obtained from IPR-stimulated mice has proved to be a highly advantageous method to evaluate salivary gland hypertrophy both at very early stages after the trophic stimulation and late after the occurrence of the trophic episode.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.     

Redox properties and acid-base equilibria of zucchini mavicyanin

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.     

Intranuclear inclusions in fine needle aspirates of bronchial low grade mucoepidermoid carcinoma with clear cell change: a report of two cases

BACKGROUND: Mucoepidermoid carcinoma of the bronchus is a rare neoplasm that can be recognized on histology as well as cytology by the presence of three characteristic cell types: mucus secreting, epidermoid and intermediate. We encountered two cases displaying unusual cytologic features, including clear intranuclear inclusions. CASES: Two females, aged 33 and 39, presented with an intrabronchial tumor and pulmonary parenchymatous mass, respectively. Fine needle aspiration of both tumors showed similar cytologic features, with a dominant population of cells with bland nuclei and wide cytoplasm, and frequent intranuclear inclusions. A minor component of mucus-secreting cells was also recognized. Histologically, both tumors corresponded to the clear cell variant of mucoepidermoid carcinoma. CONCLUSION: The cytologic picture in our cases has not been described previously in fine needle aspirates of mucoepidermoid carcinoma, in neither the bronchus nor salivary gland. The differential diagnosis of a monotonous population of epithelial cells with intranuclear inclusions involves bronchioloalveolar carcinoma, but the absence of the characteristic sheet pattern, as well as the clinical and image findings, excludes this possibility. The lack of atypia and intrabronchial location limits the scope to carcinoid and salivary gland-type tumors of the bronchus. Since we were aware of the possibility of unusual cytologic presentations of mucoepidermoid carcinomas, search for different cellular populations suggested the precise diagnosis.