RESUMO
The infectious agent for African trypanosomiasis, Trypanosoma brucei, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims interact with each other. Stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. However, TbTim10 shows weaker associations with TbTim13, which has a stronger connection with TbTim17. Each of the small TbTims also interacts strongly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial for maintaining the steady-state levels of the TbTIM17 complex. Further analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except for TbTim13, is present in ~70 kDa complexes, possibly existing in heterohexameric forms. In contrast, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionates with TbTim17. Altogether, our results demonstrate that, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific to T. brucei.
Assuntos
Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Membrana Transportadoras/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Protozoários/químicaRESUMO
Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in â¼70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei .
RESUMO
The protein translocase of the mitochondrial inner membrane in Trypanosoma brucei, TbTIM17, forms a modular complex in association with several other trypanosome-specific proteins. To identify transiently interacting proximal partner(s) of TbTim17, we used Biotinylation Identification (BioID) by expressing a modified biotin ligase-TbTim17 (BirA∗-TbTim17) fusion protein in T. brucei. BirA∗-TbTim17 was targeted to mitochondria and assembled in the TbTIM complex. In the presence of biotin, BirA∗-TbTim17 biotinylated several mitochondrial proteins. Interestingly, TbHsp84/TbTRAP1, a mitochondrial Hsp90 homolog, was identified as the highest enriched biotinylated proteins. We validated that interaction and colocalization of TbTim17 and TbHsp84 in T. brucei mitochondria by coimmunoprecipitation analysis and confocal microscopy, respectively. TbTim17 association with TbTRAP1 increased several folds during denaturation/renaturation of mitochondrial proteins in vitro, suggesting TbTRAP1 acts as a chaperone for TbTim17 refolding. We demonstrated that knockdown of TbTRAP1 reduced cell growth and decreased the levels of the TbTIM17, TbTim62, and mitochondrial (m)Hsp70 complexes. However, ATPase, VDAC, and Atom69 complexes were minimally affected. Additionally, the steady state levels of TbTim17, TbTim62, and mHsp70 were reduced significantly, but Atom69, ATPase ß, and RBP16 were mostly unaltered due to TbTRAP1 knockdown. Quantitative proteomics analysis also showed significant reduction of TbTim62 along with a few other mitochondrial proteins due to TbTRAP1 knockdown. Finally, TbTRAP1 depletion did not hamper the import of the ectopically expressed TbTim17-2xMyc into mitochondria but reduced its assembly into the TbTIM17 complex, indicating TbTRAP1 is critical for assembly of TbTim17. This is the first report showing the role of TRAP1 in the TIM complex assembly in eukaryotes.
Assuntos
Proteínas de Protozoários , Trypanosoma brucei brucei , Adenosina Trifosfatases/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
The authors wish to make the following correction to this paper [...].
RESUMO
Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8-10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.
Assuntos
Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Antimetabólitos/metabolismo , Antimetabólitos/farmacologia , Ergosterol , Humanos , Camundongos , Esteroides/farmacologia , Esteróis/metabolismo , Esteróis/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/prevenção & controleRESUMO
PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.
Assuntos
Malária Falciparum , Plasmodium falciparum , Humanos , Fosfolipídeos/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transdução de SinaisRESUMO
Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of the translocase of the mitochondrial inner membrane 50 (TbTim50). It has a pair of characteristic phosphatase signature motifs, DXDX(T/V). Here, we demonstrated that, besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. Mutations of D242 and D244, but not of D345and D347, to alanine abolished these activities. In silico structural homology models identified the putative binding interfaces that may accommodate different phosphosubstrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential (ΔΨ). TbTim50 knockdown (KD) also reduced the population of G2/M phase and increased that of G1 phase cells; inhibited segregation and caused overreplication of kinetoplast DNA (kDNA), and reduced BF cell growth. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed with upregulation of expression of a few stumpy marker genes. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice. Proteomics analysis showed reductions in levels of the translation factors, flagellar transport proteins, and many proteasomal subunits, including those of the mitochondrial heat shock locus ATPase (HslVU), which is known to play a role in regulation of kinetoplast DNA (kDNA) replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Together, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and the cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. IMPORTANCE African trypanosomiasis is a neglected tropical disease caused by the parasitic protozoan Trypanosoma brucei. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF parasites, dwelling in mammalian blood, produce ATP from glycolysis and hydrolyze ATP in mitochondria for generation of inner membrane potential. We found that TbTim50, a haloacid dehalogenase (HAD) family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced levels of CL and mitochondrial ΔΨ and caused a detrimental effect on many cellular functions. Cells accumulated in the G1 phase, and the kinetoplast was overreplicated, likely due to depletion of mitochondrial proteasome (mitochondrial heat shock locus ATPase [HslVU]), a master regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of expression of a few stumpy-specific genes. TbTim50 is essential for T. brucei survival and is an important therapeutic target for African trypanosomiasis.
Assuntos
Ciclo Celular , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Animais , Linhagem Celular , DNA de Cinetoplasto/genética , DNA de Cinetoplasto/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Fosforilação , Proteínas de Protozoários/genéticaRESUMO
Mitochondria are essential in eukaryotes. Besides producing 80% of total cellular ATP, mitochondria are involved in various cellular functions such as apoptosis, inflammation, innate immunity, stress tolerance, and Ca2+ homeostasis. Mitochondria are also the site for many critical metabolic pathways and are integrated into the signaling network to maintain cellular homeostasis under stress. Mitochondria require hundreds of proteins to perform all these functions. Since the mitochondrial genome only encodes a handful of proteins, most mitochondrial proteins are imported from the cytosol via receptor/translocase complexes on the mitochondrial outer and inner membranes known as TOMs and TIMs. Many of the subunits of these protein complexes are essential for cell survival in model yeast and other unicellular eukaryotes. Defects in the mitochondrial import machineries are also associated with various metabolic, developmental, and neurodegenerative disorders in multicellular organisms. In addition to their canonical functions, these protein translocases also help maintain mitochondrial structure and dynamics, lipid metabolism, and stress response. This review focuses on the role of Tim50, the receptor component of one of the TIM complexes, in different cellular functions, with an emphasis on the Tim50 homologue in parasitic protozoan Trypanosoma brucei.
Assuntos
Homeostase , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Transporte ProteicoRESUMO
Hydrogels are reported to have various biomedical field applications, and many reports also suggest that soft gels promote stem cell differentiation. Chondrogenic differentiation of mesenchymal stem cells (MSC) is significant in articular cartilage repair. This study focuses on polysaccharide-based hydrogels which enhance chondrocyte lineage differentiation of MSC when grown in the hydrogels. This study implies that the prepared hydrogels promote specific lineage without any external chemical induction factors. The techniques, including immunofluorescence and functional assays to assess the differentiation and in vivo implantation, were employed. All observations paved the way towards confirmation that the galactoxyloglucan-based hydrogel is an attractive candidate for supporting stem cell growth and cartilaginous differentiation.
Assuntos
Resinas Acrílicas/química , Galactose/química , Glucanos/química , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Cartilagem Articular , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Condrogênese , Hidrogéis/química , Ratos , Ratos WistarRESUMO
BACKGROUND: The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus-encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS). Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex. RESULTS: TbTim54, a trypanosome-specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited the in vitro mitochondrial import and assembly of the internal signal-containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N-terminal signal-containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate-specificity of TbTim54 for internal-signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N-terminal domain of TbTim17. CONCLUSION: TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal-containing IM proteins in T. brucei. SIGNIFICANCE: We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 in T. brucei.
Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Transporte ProteicoRESUMO
The translocases of the mitochondrial outer and inner membranes, the TOM and TIMs, import hundreds of nucleus-encoded proteins into mitochondria. TOM and TIMs are multi-subunit protein complexes that work in cooperation with other complexes to import proteins in different sub-mitochondrial destinations. The overall architecture of these protein complexes is conserved among yeast/fungi, animals, and plants. Recent studies have revealed unique characteristics of this machinery, particularly in the eukaryotic supergroup Excavata. Despite multiple differences, homologues of Tim17, an essential component of one of the TIM complexes and a member of the Tim17/Tim22/Tim23 family, have been found in all eukaryotes. Here, we review the structure and function of Tim17 and Tim17-containing protein complexes in different eukaryotes, and then compare them to the single homologue of this protein found in Trypanosoma brucei, a unicellular parasitic protozoan.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sequência Conservada , Fungos/genética , Fungos/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Plantas/genética , Plantas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Trypanosoma brucei brucei/genéticaRESUMO
Communicated by Ramaswamy H. Sarma.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Inibidores Enzimáticos , Inibidores Enzimáticos/farmacologiaRESUMO
Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.
Assuntos
Homeostase , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Adenilato Quinase/metabolismo , Mitocôndrias , Estresse Oxidativo , Fosforilação , Proteômica , Trypanosoma brucei brucei/enzimologiaRESUMO
Mechanisms by which 3'-phosphorylated phosphoinositides (3'-PIPs) regulate the development of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are poorly understood. The catabolic process of autophagy, which is dependent on autophagy-related proteins (ATGs), is one of the major targets of 3'-PIPs in yeast and mammals. In the present study, we identified autophagy-related protein ATG18 as an effector of 3'-PIPs in these parasites. Pfalciparum ATG18 (PfATG18) and Tgondii ATG18 (TgATG18) interact with 3'-PIPs but exhibited differences in their specificity of interaction with the ligand PIP. The conditional knockdown of Tgondii or Pfalciparum ATG18 (Tg/PfATG18) impaired replication of parasites and resulted in their delayed death. Intriguingly, ATG18 depletion resulted in the loss of the apicomplexan parasite-specific nonphotosynthetic plastid-like organelle apicoplast, which harbors the machinery for biosynthesis of key metabolites, and the interaction of ATG18 to phosphatidylinositol 3-phosphate (PI3P) was critical for apicoplast inheritance. Furthermore, ATG18 regulates membrane association and apicoplast localization of ATG8. These findings provide insights into a novel noncanonical role of ATG18 in apicoplast inheritance. This function of ATG18 in organelle biogenesis is unprecedented in any organism and may be conserved across most apicomplexan parasites.IMPORTANCE Typically, autophagy is a catabolic process utilized by cells for their survival upon encountering nutrient-limiting conditions. The autophagy machinery is very tightly regulated, and autophagy-related genes (ATGs) play a pivotal role in this process. In the present study, we report a novel noncanonical function of autophagy-related protein ATG18 in inheritance of the nonphotosynthetic plastid-like organelle apicoplast in apicomplexan parasites Plasmodium and Toxoplasma ATG18 depletion in these parasites resulted in "delayed death," which was the result of loss of apicoplast and impaired parasite division. Pf/TgATG18 interact with 3'-phosphorylated PIPs, which guide their cellular localization in the parasite, which is essential for their function.
Assuntos
Apicoplastos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Biogênese de Organelas , Plasmodium falciparum/metabolismo , Toxoplasma/metabolismo , Apicoplastos/genética , Autofagia , Proteínas Relacionadas à Autofagia/genética , Replicação do DNA , Técnicas de Silenciamento de Genes , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genéticaRESUMO
Second messengers such as phosphoinositides and calcium are known to control diverse processes involved in the development of malaria parasites. However, the underlying molecular mechanisms and pathways need to be unraveled, which may be achieved by understanding the regulation of effectors of these second messengers. Calcium-dependent protein kinase (CDPK) family members regulate diverse parasitic processes. Because CDPKs are absent from the host, these kinases are considered as potential drug targets. We have dissected the function of an atypical CDPK from Plasmodium falciparum, PfCDPK7. The domain architecture of PfCDPK7 is very different from that of other CDPKs; it has a pleckstrin homology domain adjacent to the kinase domain and two calcium-binding EF-hands at its N terminus. We demonstrate that PfCDPK7 interacts with PI(4,5)P2 via its pleckstrin homology domain, which may guide its subcellular localization. Disruption of PfCDPK7 caused a marked reduction in the growth of the blood stage parasites, as maturation of rings to trophozoites was markedly stalled. In addition, parasite proliferation was significantly attenuated. These findings shed light on an important role for PfCDPK7 in the erythrocytic asexual cycle of malaria parasites.
Assuntos
Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Genes de Protozoários , Fosfatidilinositol 4,5-Difosfato/metabolismo , Plasmodium falciparum/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo MensageiroRESUMO
Engineering three-dimensional (3-D) porous scaffolds with precise bio-functional properties is one of the most important issues in tissue engineering. In the present study, a three-dimensional gelatin-hyaluronic acid-alginate (GHA) polymeric composite was synthesized by freeze-drying, which was followed by ionic crosslinking using CaCl2, and evaluated for its suitability in bone tissue engineering applications. The obtained matrix showed high porosity (85%), an interconnected pore morphology and a rapid swelling behavior. The rheological analysis of GHA showed a viscoelastic characteristic, which suggested a high load bearing capacity without fractural deformation. The influence of the GHA matrix on cell growth and on modulating the differentiation ability of mesenchymal stem cells was evaluated by different biochemical and immunostaining assays. The monitoring of cells over a period of four weeks showed increased cellular proliferation and osteogenic differentiation without external growth factors, compared with control (supplemented with osteogenic differentiation medium). The in vivo matrix implantation showed higher matrix-tissue integration and cell infiltration as the duration of the implant increased. These results suggest that a porous GHA matrix with suitable mechanical integrity and tissue compatibility is a promising substrate for the osteogenic differentiation of stem cells for bone tissue engineering applications.
Assuntos
Alginatos/química , Gelatina/química , Ácido Hialurônico/química , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
Nanotechnology has become important in various disciplines of technology and science. It has proven to be a potential candidate for various applications ranging from biosensors to the delivery of genes and therapeutic agents to tissue engineering. Scaffolds for every application can be tailor made to have the appropriate physicochemical properties that will influence the in vivo system in the desired way. For highly sensitive and precise detection of specific signals or pathogenic markers, or for sensing the levels of particular analytes, fabricating target-specific nanomaterials can be very useful. Multi-functional nano-devices can be fabricated using different approaches to achieve multi-directional patterning in a scaffold with the ability to alter topographical cues at scale of less than or equal to 100 nm. Smart nanomaterials are made to understand the surrounding environment and act accordingly by either protecting the drug in hostile conditions or releasing the "payload" at the intended intracellular target site. All of this is achieved by exploiting polymers for their functional groups or incorporating conducting materials into a natural biopolymer to obtain a "smart material" that can be used for detection of circulating tumor cells, detection of differences in the body analytes, or repair of damaged tissue by acting as a cell culture scaffold. Nanotechnology has changed the nature of diagnosis and treatment in the biomedical field, and this review aims to bring together the most recent advances in smart nanomaterials.
Assuntos
Tecnologia Biomédica/métodos , Nanoestruturas/química , Animais , Técnicas Biossensoriais , Técnicas de Transferência de Genes , Humanos , Nanocápsulas/química , Nanoestruturas/ultraestrutura , Engenharia TecidualRESUMO
Bio-polymeric mosquitocidal formulations were developed for the control release of Bacillus sphaericus ISPC-8 by the immobilization of its spore-crystal complex onto the macroporous polymeric matrices. The biodegradable formulations were synthesized at sub-zero temperature using natural polymeric substrates like agarose, alginate, cellulose, non-adsorbent cotton, wooden cork powder and also magnetite nanoparticles. The obtained polymeric matrices were morphologically characterized, which showed 85-90% porosity, uniform pores distribution, high permeability and controlled degradation (19-30%) in 4 weeks depending upon the composition of formulations. Further, the polymeric macroporous formulations were tested for persistence of mosquitocidal activity against Culex quinquefasciatus larvae. Unformulated B. sphaericus ISPC-8 spores retained 54% of larvicidal activity after 7 days, which completely reduced after 35 days of treatment. However, the immobilized B. sphaericus spores in agarose-alginate formulations showed high larvicidal activity on day 7 and retained about 45% activity even after 35 days of treatments. Studies on UV-B and pH dependent inactivation of toxins and spore viability showed that these formulations were significantly protecting the spores as compared to the unformulated spores, which suggest its potential application for the mosquito control program.
Assuntos
Bacillus/fisiologia , Culex , Controle Biológico de Vetores/métodos , Animais , Bacillus/efeitos da radiação , Bacillus/ultraestrutura , Toxinas Bacterianas/administração & dosagem , Biopolímeros/química , Células Imobilizadas , Química Farmacêutica , Preparações de Ação Retardada , Concentração de Íons de Hidrogênio , Insetos Vetores , Larva , Teste de Materiais , Microscopia Eletrônica de Varredura , Porosidade , Esporos Bacterianos/fisiologia , Esporos Bacterianos/efeitos da radiação , Esporos Bacterianos/ultraestrutura , Raios UltravioletaRESUMO
Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin-laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.