Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

<p>Abstract</p> <p>Background</p> <p>Tuberculosis is an infectious bacterial disease in humans caused primarily by <it>Mycobacterium tuberculosis</it>, and infects one-third of the world's total population. <it>Mycobacterium bovis </it>bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated <it>M. bovis </it>BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions.</p> <p>Results</p> <p>Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work.</p> <p>Conclusions</p> <p>In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction.</p

DETC Induces Leishmania Parasite Killing in Human In Vitro and Murine In Vivo Models: A Promising Therapeutic Alternative in Leishmaniasis

Background: Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. Methodology/Principal Findings: We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p,0.001) and is able to ??????sterilize?????? Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p,0.001) and parasite destruction (p,0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p,0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. Conclusions/Significance: Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

BACKGROUND: Casein kinase 1 delta (CK1delta) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1delta has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1delta in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining of CK1delta was performed using three different antibodies against CK1delta. A high expression of CK1delta was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. CONCLUSIONS: These results give an overview of the cell-type specific expression of CK1delta in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1delta, where CK1delta can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1delta in these tissues

Adverse effects of the antimalaria drug, mefloquine: due to primary liver damage with secondary thyroid involvement?

BACKGROUND: Mefloquine is a clinically important antimalaria drug, which is often not well tolerated. We critically reviewed 516 published case reports of mefloquine adverse effects, to clarify the phenomenology of the harms associated with mefloquine, and to make recommendations for safer prescribing. PRESENTATION: We postulate that many of the adverse effects of mefloquine are a post-hepatic syndrome caused by primary liver damage. In some users we believe that symptomatic thyroid disturbance occurs, either independently or as a secondary consequence of the hepatocellular injury. The mefloquine syndrome presents in a variety of ways including headache, gastrointestinal disturbances, nervousness, fatigue, disorders of sleep, mood, memory and concentration, and occasionally frank psychosis. Previous liver or thyroid disease, and concurrent insults to the liver (such as from alcohol, dehydration, an oral contraceptive pill, recreational drugs, and other liver-damaging drugs) may be related to the development of severe or prolonged adverse reactions to mefloquine. IMPLICATIONS: We believe that people with active liver or thyroid disease should not take mefloquine, whereas those with fully resolved neuropsychiatric illness may do so safely. Mefloquine users should avoid alcohol, recreational drugs, hormonal contraception and co-medications known to cause liver damage or thyroid damage. With these caveats, we believe that mefloquine may be safely prescribed in pregnancy, and also to occupational groups who carry out safety-critical tasks. TESTING: Mefloquine's adverse effects need to be investigated through a multicentre cohort study, with small controlled studies testing specific elements of the hypothesis