Functional characterization of an uncoupling protein in goldfish white skeletal muscle

Uncoupling proteins (UCP) are able to increase H⁺ leakage across the inner mitochondrial membrane, thus dissipating the membrane potential and increasing oxygen consumption. Despite the identification of several UCP orthologs in birds, reptiles, amphibians and fish, little is known about their functional properties in fish. The aim of this work was to identify and characterize a UCP in mitochondria found in goldfish white skeletal muscle. Western blot analysis, using a polyclonal antibody raised against mammalian UCP3, showed a single band at approximately 32 kDa. During non-phosphorylating respiration, we observed that palmitate promoted a dose-dependent increase in oxygen consumption that is abolished by addition of BSA (fatty acid chelator). Interestingly, this palmitate-induced increase in oxygen consumption was not inhibited by GDP, a well-known UCP inhibitor. In phosphorylating mitochondria, palmitate lowered both ADP/O ratio (number of atoms of phosphorus incorporated as ATP per molecule of O₂ consumed) and the respiratory control ratio. Moreover, we found that different fatty acids can modulate mitochondrial membrane potential. In conclusion, our results suggest that goldfish UCP is functionally similar to the UCP found in other species, including mammals.     

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ?leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ?leuD strain. The inhibitory effect of BCG ?leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ?leuD/Ag85B treatment was evaluated by Western blotting. BCG ?leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ?leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ?leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ?leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.     

M2-Polarized Macrophages Determine Human Cutaneous Lesions in Lacaziosis

Lacaziosis is a cutaneous chronic mycosis caused by Lacazia loboi. Macrophages are important cells in the host immune response in fungal infections. The macrophage population exhibits strong plasticity that varies according to the stimuli in the microenvironment of lesions M1 profile promotes a Th1 pattern of cytokines and a microbicidal function and M2 is related to Th2 cytokines and immunomodulatory response. We investigated the population of M1 and M2 polarized macrophages in human cutaneous lesions. A total of 27 biopsies from human lesions were submitted to an immunohistochemistry protocol using antibodies to detect M1 and M2 macrophages (Arginase-1, CD163, iNOS, RBP-J and cMAF). We could observe high number of cells expressing Arginase1, CD163 and c-MAF that correspond to elements of the M2 profile of macrophage, over iNOS and RBP-J (elements of the M1 profile). The results suggest a predominant phenotype of M2 macrophages, which have an immunomodulatory role and probably contributing to chronicity of Lacaziosis.

     

Characterization of the immunogenic and antigenic potential of putative lipoproteins from Leptospira interrogans

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.     

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.     

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by florescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8–10% using naked DNA or liposomes to 40–45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.     

Sequence diversity at the proximal 14q32.1 SERPIN subcluster: evidence for natural selection favoring the pseudogenization of SERPINA2

The superfamily of serine protease inhibitors (SERPINs) plays a key role in controlling the activity of proteinases in diverse biological processes. alpha1-antitrypsin (SERPINA1), the most studied member of this family, is encoded by a gene located within the proximal 14q32.1 SERPIN subcluster, together with the highly homologous alpha1-antitrypsin-like sequence (SERPINA2), which was previously proposed to be a pseudogene. Here, we performed a resequencing study encompassing both SERPINA1 and SERPINA2 as well as the adjacent gene coding for corticosteroid-binding globulin (SERPINA6) in samples from Europe and West Africa. In the African sample, we found that a common haplotype carrying a 2-kb deletion in the SERPINA2 gene is associated with remarkable long-range homozygozity as if it was quickly driven to high frequency by natural selection acting on an advantageous variant. An analysis of the HapMap Phase I data for the Yoruba sample confirmed that variation in this subcluster carries a strong signal of positive selection. We also show that the SERPINA2 gene is expressed and probably encodes a functional SERPIN. Finally, comparisons with orthologous sequences in nonhuman primates showed that SERPINA2 is present in some great apes, but in chimpanzees it was lost by a deletion event independent from that observed in humans. In agreement with the "less is more" hypothesis, we propose that loss of SERPINA2 is an ongoing process associated with a selective advantage during recent primate evolution, possibly because of a role in fertility or in host-pathogen interactions.     

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.     

Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5‐kinase (PIKfyve) that converts phosphatidylinositol 3‐phosphate [PI(3)P] into phosphatidylinositol 3,5‐bisphosphate [PI(3,5)P₂] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non‐pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P₂ effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P₂‐dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.