Development of a Real-Time PCR for detection of <i>Mycoplasma agalactiae</i> in bulk tank milk samples and epidemiology of infection in Sardinia

In this work the Mycoplasma agalactiae p48 gene was used as a diagnostic marker for contagious agalactia (CA) of sheep and goats by Real-Time PCR. The p48 gene encodes an invariable, constantly expressed, immunodominant surface lipoprotein belongs to the basic membrane protein family. The Real-Time PCR test based on p48 resulted specific and sensible. The test performance were evaluated on bulk tank milk samples collected from 1064 ovine and 66 goat farms in sardinian region. 4.8% of sheep farms and 4.5 % of goat farms tested positive. Our results showed that the test based on the p48 gene can be used on bulk tank milk for detection and epidemiological surveillance of Mycoplasma agalactiae infections

Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells

Background. While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. Methodology/Principal Findings. Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance. Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products

May band 3 hyper-phosphorylation have a functional role in microcyte formation in heterozygous thalassemias?

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May band 3 hyper-phosphorylation have a functional role in microcyte formation in heterozygous thalassemias?

Heterozygous alpha and beta thalassemia are extremely frequent in malaria endemic areas displaying a well balanced hematological situation with very mild anemia and microcytic red cells. There is widespread consensus that thalassemia determines resistance to severe malaria although the molecular basis of the mechanism of resistance is not understood. A few studies indicate that the thalassemic cell environment does not directly harm the parasites but apparently induces the loss of viability of infected red cells and their enhanced removal by macrophages. The difficulty to develop an hypothesis on the mechanism of malaria resistance largely derives from the substantial lack of knowledge on the physiology of thalassemic red cells in heterozygotes. Although causing only mild and asymptomatic anemia, the genetic defect causes unbalanced hemoglobin chain production. It is not understood how this large excess of globin chains can be removed without effecting the viability of the red cell. It is generally accepted that the loss of red cell membrane leading to microcytosis may represent the counterpart of hemoglobin chain excess removal but the mechanisms underlying this process are not defined. Our previous observations showed that in splenectomized intermediate thalassemia patients the Hb increase (from 7.7 to 9.1 g/dl) in comparison to non-splenectomized patients was due to a net increase of mean MCV (from 71 to 82 fl) and not to an increased number of red cells. This clinical evidence is not, therefore, in accordance with the concept that, in thalassemia, the spleen mainly acts to remove altered red cells but suggests that the spleen has an active role in reducing red cell volume. We have observed a larger amount of hemichromes bound to the red cell membrane in splenectomised patients. Those data are an indication that splenic macrophages exert a role in removing the hemichromes leading to a decrease of red cell volume. Histological studies show the role of splenic macrophages in “pitting” part of the red cell membrane containing hemichromes. Based on some recently published results and some preliminary data we developed the hypothesis that a physiological mechanism may permit the removal of abnormal globins deposited on the cytoplasmic side of the red cell membrane. This process should require a series of sequential events: a) binding of denatured globin chains to specific membrane protein ligands b) selective modification of the ligand bound to the globins to induce its clustering and the accumulation of all globin excess in a limited area of the membrane c) some mechanism leading to the recognition and binding of the clustered globin ligand by macrophages d) weakening of membrane structure in the area of denatured globin accumulation to allow its selective removal by macrophages and/or vesiculation of locally destabilized areas without interfering with the overall integrity of red cell membrane. This mechanism should allow globin excess removal minimizing the area of the membrane to be sacrificed therefore obtaining smaller but still viable biconcave red cells

Contact-dependent disruption of the host cell membrane skeleton induced by <i>Trichomonas vaginalis</i>

This report presents evidence showing that the pathogenetic process of the protozoan parasite Trichomonas vaginalis involves degradation of the target cell membrane skeleton; spectrin, the most representative protein within this structure, has been identified as the main molecular target. Degradation of the target cell spectrin is accomplished only upon contact with the parasite, and immunochemical and immunofluorescence studies performed with the erythrocyte as a model demonstrate that degradation of the protein takes place before target cell lysis. A preliminary characterization of the effectors involved has led to the identification of a nonsecreted 30-kDa proteinase which is characterized by a high specificity for spectrin. This molecule is suggested as the main effector responsible for cytoskeletal disruption

A 2DE approach for high-throughput antigen separation applicable to mAb production

A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein–protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development ofmAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 g of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available formonoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the highthroughput monoclonal antibody process providing a vast array of high quality antigens at very low cost

Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells

Background: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. Methodology/Principal Findings: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation