Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

 Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue. Received: 23 December 1999 / Accepted: 3 June 2000     

Isolation from a Shea Cake Digester of a Tannin-Tolerant Escherichia coli Strain Decarboxylating p-Hydroxybenzoic and Vanillic Acids

A facultatively anaerobic, mesophilic, Gram-negative, non-motile, non-sporulated bacterium, designated strain C2, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, after enrichment on tannic acid. The straight rods occurred singly or in pairs. Strain C2 fermented numerous carbohydrates (fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose) and peptides (Biotrypcase, Casamino acids, and yeast extract), producing acid and gas, and had a G + C content of 51.6 ± 0.1 mol %. Strain C2 was very closely related to Escherichia coli (= DSM 30083^T) phylogenetically (similarity of 99%), genotypically (DNA homology of 79%), and phenotypically. The isolate tolerated tannic acid (hydrolyzable tannin) and decarboxylated by non-oxidative decarboxylation only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaicol, under anaerobic and aerobic conditions without further degradation. Adding glucose increased growth and the rate of conversion. High concentrations of p-hydroxybenzoic acid or vanillic acid inhibited growth, and decarboxylation could not occur completely, suggesting phenol toxicity. In contrast, the type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added. Received: 30 July 2001 / Accepted: 17 August 2001     

Respiratory variations of aortic VTI: a new index of hypovolemia and fluid responsiveness

In 12 mechanically ventilated and anesthetized rabbits, we investigated whether the magnitude of respiratory changes in the aortic velocity time integral (VTI(Ao)), recorded by transthoracic echocardiography (TTE) during a stepwise blood withdrawal and restitution, could be used as a reliable indicator of volume depletion and responsiveness. At each step, left and right ventricular dimensions and the aortic diameter and VTI(Ao) were recorded to calculate stroke volume (SV) and cardiac output (CO). Respiratory changes of VTI(Ao) (maximal - minimal values divided by their respective means) were calculated. The amount of blood withdrawal correlated negatively with left and right ventricular diastolic diameters, VTI(Ao), SV, and CO and correlated directly with respiratory changes of VTI(Ao). Respiratory VTI(Ao) variations (but not other parameters) at the last blood withdrawal step was also correlated with changes in SV after blood restitution (r = 0.83, P < 0.001). In conclusion, respiratory variations in VTI(Ao) using TTE appear to be a sensitive index of blood volume depletion and restitution. This dynamic parameter predicted fluid responsiveness more reliably than static markers of cardiac preload.     

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.     

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.     

Conjugated linoleic acid isomers in mitochondria: evidence for an alteration of fatty acid oxidation

The beneficial effects exerted by low amounts of conjugated linoleic acids (CLA) suggest that CLA are maximally conserved and raise the question about their mitochondrial oxidizability. Cis-9,trans-11-C(18:2) (CLA1) and trans-10,cis-12-C(18:2) (CLA2) were compared to cis-9,cis-12-C(18:2) (linoleic acid; LA) and cis-9-C(16:1) (palmitoleic acid; PA), as substrates for total fatty acid (FA) oxidation and for the enzymatic steps required for the entry of FA into rat liver mitochondria. Oxygen consumption rate was lowest when CLA1 was used as a substrate with that on CLA2 being intermediate between it and the respiration on LA and PA. The order of the radiolabeled FA oxidation rate was PA > LA > CLA2 > CLA1. Transesterification to acylcarnitines of the octadecadienoic acids were similar, while uptake across inner membranes of CLA1 and, to a lesser extent, of CLA2 was greater than that of LA or PA. Prior oxidation of CLA1 or CLA2 made re-isolated mitochondria much less capable of oxidising PA or LA under carnitine-dependent conditions, but without altering the carnitine-independent oxidation of octanoic acid. Therefore, the CLA studied appeared to be both poorly oxidizable and capable of interfering with the oxidation of usual FA at a step close to the beginning of the beta-oxidative cycle.     

Role of two dileucine-like motifs in insulin receptor anchoring to microvilli

In the absence of ligand, the insulin receptor is maintained on microvilli on the cell surface. A dileucine motif (LL(986-987)) is necessary but not sufficient for this anchoring, which also required the presence of additional sequence(s) downstream of position 1000. The aim of the present study was to identify this (these) additional sequence(s). First, exons 16 or 17 were fused to the extracellular and transmembrane domains of complement receptor 1 and stably expressed in Chinese hamster ovary cells. Results obtained indicate that exon 17 is sufficient for anchoring to microvilli. Second, analysis of insulin receptor mutants truncated within exon 17 demonstrated that whereas receptors truncated at position 1000 showed no preferential association with microvilli, receptors truncated at position 1012 displayed a level of association identical to that of the full-length insulin receptor. Third, mutation of a diisoleucine motif (II(1006-1007)) present within this 12-amino acid stretch abrogated the preferential association of the receptor with microvilli. These results indicate that the domain required for association of insulin receptor with microvilli is contained within the region encoded by exon 17 and that, within this sequence, two dileucine-like motifs (LL(986-987) and II(1006-1007)) play a crucial role.     

B cell positive selection by soluble self-antigen

It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection.     

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.