Vaginal bacterial microflora modifications during the growth of healthy cows

The aim of this work was first, to determine the predominant groups capable of colonizing the vagina and maintaining high numbers with time. The normal microbial flora of the cow's vagina and its evolution from weaning to service was then studied using standard microbiological methods. The results show that the most dominant bacteria belong to the streptococci, followed by the staphylococci, with similar levels during the whole study period. Enterobacteriaceae and lactobacilli were present at very low levels, the latter increasing during the cow's growth, suggesting some kind of hormonal influence. The results will allow the selection of micro-organisms with probiotic characteristics, classified as GRAS (Generally Regarded as Safe), to be used in the prevention of infections in the vaginal tract of cows, such as metritis, which produces delayed periods between partum and conception, and consequent economic losses.     

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.     

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.     

The Rhizobium etli rpoN Locus: DNA Sequence Analysis and Phenotypical Characterization of rpoN, ptsN, and ptsA Mutants

The rpoN region of Rhizobium etli was isolated by using the Bradyrhizobium japonicum rpoN1 gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258, rpoN, ORF191, and ptsN, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and ptsN are homologous to conserved ORFs found downstream from rpoN genes in other bacterial species. Unlike in most other microorganisms, rpoN and ORF191 are separated by approximately 1.6 kb. The R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of nifH, and the metabolism of C₄-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the rpoN gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the ptsN gene resulted in a decrease of melanin synthesis and nifH expression. In a search for additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in ptsA, a gene homologous to the Escherichia coli gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA mutant also displayed reduced expression of nifH. The ptsN and ptsA mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C₄-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that ptsN and ptsA are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.     

Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants

For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and F_ab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and F_abfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeF_ab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or F_ab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream. ⁴Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be     

Baseline NT-ProBNP level predicts success of cardioversion of atrial fibrillation with flecainide

BackgroundPatients with acute-onset symptomatic atrial fibrillation (AF) can be treated with flecainide. However, flecainide may induce arrhythmias and/or exaggerate heart failure. Therefore, validated markers to predict the efficacy of flecainide and prevent adverse effects are required. We hypothesised that lower NT-proBNP plasma levels correlate with higher success rates of cardioversion with flecainide in patients with AF.MethodsIn this prospective single-centre study, we included 112 subsequent patients with acute-onset (< 24 h) symptomatic AF. Patients with symptoms of heart failure and ECG signs of ischaemia were excluded. Baseline laboratory measurements, including NT-proBNP, were done. Echocardiograms were performed ~ 2 weeks after restoration of SR.ResultsCardioversion with flecainide was successful in 91 patients (87 %). NT-proBNP was lower in patients with successful cardioversion (P < 0.001). Logistic regression indicated NT-proBNP as an independent predictor of successful cardioversion. A cut-off NT-proBNP value of 1550 pg/ml provided optimal test accuracy to predict successful cardioversion.ConclusionIn patients with < 24 h of symptomatic AF, NT-proBNP levels up to 1550 pg/ml correlate with high success rates (94 %) of cardioversion with flecainide. Conversely, NT-proBNP higher than 1550 pg/ml correlates with poor success rates (36 %). Further research is needed to validate the predictive value of NT-proBNP for successful cardioversion with flecainide.     

The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.Membrane transporters play essential roles in cellular homeostasis by importing substrates critical for cell growth and differentiation or by exporting substrates that cause toxicity. There are five major categories of membrane transporters consisting of over 550 transporter superfamilies (41). The major facilitator superfamily (MFS) is the largest and most diverse superfamily, consisting of over 10,000 members (31, 41). Transporters in this superfamily consist of 12 to 14 transmembrane (TM)-spanning segments and transport substrates as diverse as sugars, polyols, drugs, neurotransmitters, amino acids, organic/inorganic ions, and peptides (31). Recently, a disruption of MFS transporters that is associated with human diseases has been described, further confirming their role in the maintenance of normal cell homeostasis. The DIRC2 MFS transporter (substrate transported unknown) is disrupted in renal cell carcinoma cosegregating with a t(2;3)(q35;q21) chromosomal translocation (4). Mutations in the thiamine transporter THTR1 have been shown to be responsible for Rogers syndrome (14, 21), a thiamine-responsive megaloblastic anemia. We have recently reported that a disruption in the heme exporter FLVCR1 (MFSD7B) plays a role in Diamond Blackfan anemia (DBA) (40), a fatal infant anemia characterized by a block in erythroid progenitor cell development (3, 12, 13). The abrogation of FLVCR1 function in primary human hematopoietic stem cells (40) or in a human erythroid cell line (37) specifically disrupts erythropoiesis, mimicking the hematological features observed for patients with DBA. We have reported previously that FLVCR1 is disrupted not as a consequence of mutations in the FLVCR1 coding region but due to the aberrant splicing of specific FLVCR1 exons that reduces the expression and cell surface localization of the encoded FLVCR1 protein (40). Interestingly, the THTR1 and FLVCR1 proteins were shown previously to function as receptors for entry by feline leukemia retrovirus (FeLV) subgroup A (FeLV-A) (25) and FeLV-C (36, 46), respectively. These viruses disrupt the cellular function of these proteins in infected cats and can induce diseases that correlate with Rogers syndrome (17) and DBA (1, 28).Recently, mutations in the cell surface protein FLVCR2 (MFSD7C), an MFS transporter member, have been shown to be associated with Fowler syndrome (22, 26), a proliferative vascular disorder of the brain (16). A previous study (6) suggested that FLVCR2 functions as a calcium-chelate transporter based on its expression in murine and human tissues involved in calcium homeostasis. We have shown previously that FLVCR2 is highly related to the heme exporter/retroviral receptor FLVCR1 (7), and we have recently shown it to function as a receptor for the subgroup C FeLV variant FY981 (42). Based on its close sequence relationship to the heme exporter/retroviral receptor FLVCR1 and based on previous reports showing that retroviruses often adapt to use closely related cell surface proteins as receptors for infection (27, 30, 44), we investigated the heme transport function of FLVCR2. Here, we show the physiological function of FLVCR2 as an importer of heme.