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A new Co (II) complex incorporating a novel Schiff base ligand acquired from the condensation of 3,3'-Methylenedianiline and 2-Hydroxy-5-bromobenzaldehyde was synthesized and characterized. The synthesized complex was air and moisture stable, monomeric, and non-electrolytic in nature. Based on physical and spectral studies, tetrahedral conformation was ascribed to the synthesized Co (II) complex.Density Functional Theory (DFT) was used to analysis different electronic parameters of the optimized structure of Co(II) complex to reveal its stability.Using different analytic and spectroscopic techniques, the new Co (II) complex was established to interact with DNA quite effectively and works as an efficient metallo intercalators. The synthesized complex was discovered to cleave DNA significantly, so it can be inferred that the complex will inhibit the growth of pathogens. Molecular docking was performed to check the binding affinity of the cobalt complex with different receptors, responsible for different diseases. Proteins like progesterone receptor and induced myeloid leukemia cell differentiation Mcl-1 protein showed high binding affinity with this complex, and hence the complex might have some implications for inhibition of progesterone hormones in biological systems. Biological activity of the Co (II) complex was also predicted through computational analysis with SwissADME.Using strains of Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus, an in vitro antibacterial activity of the ligand and Co (II) complex was carried out. This activity was further validated by a molecular docking investigation., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Condensing 2-phenoxyaniline with 5-chlorosalicyldehyde under reflux conditions, a 4-chloro-2-(((2-phenoxyphenyl)imino)methyl)phenol Schiff base has been Synthesized. A zinc complex was synthesized by combining the ligand in a 1:1 molar ratio with zinc sulphateheptahydrate. Mass spectroscopy, NMR, infrared, and elemental analysis were used to characterize the ligand and zinc complex. By measuring the molar conductance, the non-electrolytic character of the complex was confirmed. The zinc ion is coordinated in a pentadentate manner, according to an IR and NMR investigation. Viscosity measurements, absorption and fluorescence spectroscopy were utilized to examine the complex's interaction with CT (calf thymus) DNA. Furthermore, the ligand and complex's ADMET characteristics were ascertained through the use of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) study. Calculation of the different electronic parameters of the optimized structure through Density Functional Theory (DFT) indicated the stability of the Zn(II) complex. Molecular docking study reflected the future opportunity for the consideration of Zn(II) complex to fight against Alzheimer and Glaucoma diseases., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel metal complex was synthesized using freshly prepared 2-Amino-5-nitro-N-[(E)-thiophen-2-yl-methylidene]aniline ligand with Zn (II) sulphate heptahydrate in a 1:1 molar ratio. The ligand and the complex were characterized using different spectroscopic techniques, and the complex was assigned a distorted square pyramidal geometry. Additionally, DNA binding assays and antibacterial activity were used to assess the biological perspectives for the synthesized complex, including the ligand and complex which was further confirmed by molecular docking. Fluorescence Spectroscopy, viscosity measurement, and adsorption measurement were used to investigate the interaction of the Zn (II) complex with CT-DNA. A comparative in vitro antibacterial activity study against Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus strains were studied with free ligand and Zn (II) metal complex. The stable geometry of the complex was additionally established through computational simulation utilizing density functional theory, which was followed by the calculation of several electronic properties. The ADMET characteristics of the complex and ligand were also assessed using ADMET analysis. The in-silico ADMET properties pointed to a significant drug-likeness feature in the synthesized compounds, based on the Lipinski criteria., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

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 EC 50 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.

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 D 242 and D 244 , but not of D 345 and D 347 , 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 G 2 /M phase and increased that of G 1 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 G 1 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.

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., (© 2020 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

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.

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. brucei IMPORTANCE Trypanosoma 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 ., (Copyright © 2019 Tripathi et al.)

The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei , the protozoan parasite that causes African trypanosomiasis, possesses multiple small Tim proteins that include homologues of T. brucei Tim9 (TbTim9) and Tim10 (TbTim10) and a unique small Tim that shares homology with both Tim8 and Tim13 (TbTim8/13). Here, we found that these three small TbTims are expressed as soluble mitochondrial intermembrane space proteins. Coimmunoprecipitation and mass spectrometry analysis showed that the small TbTims stably associated with each other and with TbTim17, the major component of the mitochondrial inner membrane translocase in T. brucei Yeast two-hybrid analysis indicated direct interactions among the small TbTims; however, their interaction patterns appeared to be different from those of their counterparts in yeast and humans. Knockdown of the small TbTims reduced cell growth and decreased the steady-state level of TbTim17 and T. brucei ADP/ATP carrier (TbAAC), two polytopic mitochondrial inner membrane proteins. Knockdown of small TbTims also reduced the matured complexes of TbTim17 in mitochondria. Depletion of any of the small TbTims reduced TbTim17 import moderately but greatly hampered the stability of the TbTim17 complexes in T. brucei Altogether, our results revealed that TbTim9, TbTim10, and TbTim8/13 interact with each other, associate with TbTim17, and play a crucial role in the integrity and maintenance of the levels of TbTim17 complexes. IMPORTANCE Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite's mitochondrion represents a useful source for potential chemotherapeutic targets. Similarly to yeast and humans, mitochondrial functions depend on the import of proteins that are encoded in the nucleus and made in the cytosol. Even though the machinery involved in this mitochondrial protein import process is becoming clearer in T. brucei , a comprehensive picture of protein complex composition and function is still lacking. In this study, we characterized three T. brucei small Tim proteins, TbTim9, TbTim10, and TbTim8/13. Although the parasite does not have the classical TIM22 complex that imports mitochondrial inner membrane proteins containing internal targeting signals in yeast or humans, we found that these small TbTims associate with TbTim17, the major subunit of the TbTIM complex in T. brucei , and play an essential role in the stability of the TbTim17 complexes. Therefore, these divergent proteins are critical for mitochondrial protein biogenesis in T. brucei ., (Copyright © 2018 Smith et al.)

Trypanosoma brucei Tim17(TbTim17), the single member of the Tim17/23/22 protein family, is an essential component of the translocase of the mitochondrial inner membrane (TIM). In spite of the conserved secondary structure, the primary sequence of TbTim17, particularly the N-terminal hydrophilic region, is significantly divergent. In order to understand the function of this region we expressed two N-terminal deletion mutants (Δ20 and Δ30) of TbTim17 in T. brucei. Both of these mutants of TbTim17 were targeted to mitochondria, however, they failed to complement the growth defect of TbTim17 RNAi cells. In addition, the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria were not restored by expression of the N-terminal deletion mutants but complemented by knock-in of the full-length protein. Further analysis revealed that Δ20-TbTim17 and Δ30-TbTim17 mutants were not localized in the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by two-dimensional Blue-native/SDS-PAGE revealed that none of these mutants are assembled into the TbTim17 protein complex. However, FL-TbTim17 was integrated into the mitochondrial inner membrane and assembled into TbTim17 complex. Co-immunoprecipitation analysis showed that unlike the FL-TbTim17, mutant proteins are not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N-terminal domain of TbTim17 plays unique and essential roles for its sorting and assembly into the TbTim17 protein complex., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15±5μM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24β-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (k cat /k inact =0.13min -1 /0.08min -1 ; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC-MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases., (Copyright © 2016. Published by Elsevier B.V.)

Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17-2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae. We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Trypanosoma brucei, the causative agent for African trypanosomiasis, possesses a single mitochondrion that imports hundreds of proteins from the cytosol. However, the parasite only possesses a few homologs of the canonical protein translocases found in fungi and animals. We recently characterized a homolog of the translocase of the mitochondrial inner membrane, Tim50, in T. brucei. TbTim50 knockdown (KD) moderately reduced cell growth, decreased the mitochondrial membrane potential, and inhibited import of proteins into mitochondria. In contrast to Tim50 KD, we show here that TbTim50 overexpression (OE) increased the mitochondrial membrane potential as well as increased the production of cellular reactive oxygen species (ROS). Therefore, TbTim50 OE also inhibits cell growth. In addition, TbTim50 OE and KD cells showed different responses upon treatment with H2O2. Surprisingly, TbTim50 KD cells showed a greater tolerance to oxidative stress. Further analysis revealed that TbTim50 KD inhibits transition of cells from an early to late apoptotic stage upon exposure to increasing concentrations of H2O2. On the other hand TbTim50 OE caused cells to be in a pro-apoptotic stage and thus they underwent increased cell death upon H2O2 treatment. However, externally added H2O2 similarly increased the levels of cellular ROS and decreased the mitochondrial membrane potential in both cell types, indicating that tolerance to ROS is mediated through induction of the stress-response pathway due to TbTim50 KD. Together, these results suggest that TbTim50 acts as a stress sensor and that down regulation of Tim50 could be a survival mechanism for T. brucei exposed to oxidative stress., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.

Mitochondrial outer membrane (MOM) proteins in parasitic protozoa like Trypanosoma brucei are poorly characterized. In fungi and higher eukaryotes, Tob55 is responsible for the assembly of β-barrel proteins in the MOM. Here we show that T. brucei Tob55 (TbTob55) has considerable similarity in its primary and secondary structure to Tob55 from other species. TbTob55 is localized in T. brucei MOM and is essential for procyclic cell survival. Induction of Tob55 RNAi decreased the level of the voltage-dependent anion channel (VDAC) within 48 h. Although the primary effect is on VDAC, induction of TbTob55 RNAi for 96 h or more also decreased the levels of other nucleus encoded mitochondrial proteins. In addition, the mitochondrial membrane potential was reduced at this later time point possibly due to a reduction in the level of the proteins involved in oxidative phosphorylation. However, mitochondrial structure was not altered due to depletion of Tob55. In vitro protein import of VDAC into mitochondria with a 50-60% reduction of TbTob55 was reduced about 40% in comparison to uninduced control. In addition, the import of presequence-containing proteins such as, cytochrome oxidase subunit 4 (COIV) and trypanosome alternative oxidase (TAO) was affected by about 20% under this condition. Depletion of VDAC levels by RNAi did not affect the import of either COIV or TAO. Furthermore, TbTob55 over expression increased the steady state level of VDAC as well as the level of the assembled protein complex of VDAC, suggesting that similar to other eukaryotes TbTob55 is involved in assembly of MOM β-barrel proteins and plays an indirect role in the biogenesis of mitochondrial preproteins destined for the mitochondrial inner membrane., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a beta-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.

Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome database, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6-7-fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei.

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis in human and domestic animals, adapt in various environments during their digenetic life cycle. In this study, we found that Hsp90 is crucial for the survival of this parasite. Inhibition of Hsp90 activity by geldanamycin (GA) reduced cell growth and increased the level of Hsp90. Both the bloodstream and procyclic forms of T. brucei showed a several-fold greater sensitivity than the mammalian cells to GA and also to 17-AAG, a less toxic derivative of GA, suggesting that Hsp90 could be a potential chemotherapeuric target for African trypanosomiasis. T. brucei Hsp90 interacts with the protein phosphatase 5 (PP5) in vivo. Under normal growth conditions, T. brucei PP5 (TbPP5) and Hsp90 are primarily localized in the cytosol. However, with increase in growth temperature and GA treatment, these proteins translocate to the nucleus. Overproduction of TbPP5 by genetic manipulation reduced the growth inhibitory effect of GA, while knockdown of TbPP5 reduced cell growth more in the presence of GA, as compared to parental control. Depletion of TbPP5, however, did not prevent the induction of Hsp90 protein level during GA treatment. Together, these results suggest that TbPP5 positively regulates the function of Hsp90 to maintain cellular homeostasis during proteotoxic stresses in T. brucei.

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation., ((c) 2007 Wiley-Liss, Inc.)

The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the P13K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

Purpose: The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway and the heat shock protein family are up-regulated in multiple myeloma and are both regulators of the cyclin D/retinoblastoma pathway, a critical pathway in multiple myeloma. Inhibitors of mTOR and HSP90 protein have showed in vitro and in vivo single-agent activity in multiple myeloma. Our objective was to determine the effects of the mTOR inhibitor rapamycin and the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on multiple myeloma cells., Experimental Design: Multiple myeloma cell lines were incubated with rapamycin (0.1-100 nmol/L) and 17-AAG (100-600 nmol/L) alone and in combination., Results: In this study, we showed that the combination of rapamycin and 17-AAG synergistically inhibited proliferation, induced apoptosis and cell cycle arrest, induced cleavage of poly(ADP-ribose) polymerase and caspase-8/caspase-9, and dysregulated signaling in the phosphatidylinositol 3-kinase/AKT/mTOR and cyclin D1/retinoblastoma pathways. In addition, we showed that both 17-AAG and rapamycin inhibited angiogenesis and osteoclast formation, indicating that these agents target not only multiple myeloma cells but also the bone marrow microenvironment., Conclusions: These studies provide the basis for potential clinical evaluation of this combination for multiple myeloma patients.

Glial progenitors from the brain of normal adult Sprague-Dawley rats were compared to their initiated and malignant counterparts that were isolated from apparently normal brains of animals exposed to methylnitrosourea (MNU). Fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor (PDGF)-A or -B induced differentiation of normal progenitors to a pro-astrocytic or oligodendrocytic morphology, respectively, whereas the combination of these factors resulted in their terminal differentiation to oligodendrocytes and senescence. In contrast, initiated progenitors did not exit the cell cycle when stimulated with PDGF and/or FGF-2. cDNA oligoarray analysis and RT-PCR verification showed an early upregulation/ induction of growth factor/receptors, PDGF-A, PDGFR-beta, IGFR-1, IGF-1 and -2, IL-6, MEGF-5, FRAG-1, IRS-2, HSPG, and FGFR-1, followed by a late increase in the expression IGFBP-6, PDGF-alpha, FGFR-4A, c/ERB-A, and FGFR-4, 2, and 1 during the tumorigenic progression. Western blot analyses demonstrated that MNU exposure caused progressive reduction of p21 protein levels, an increase of Rb phosphorylation, activation of AKT and CDK2, and upregulation of FGF receptors. Double immunofluorescence labeling showed progressive increase in nuclear colocalization of FGFR1, 2, and 4, which peaked in malignant lines. It is postulated that transition of normal rat glial progenitors to an initiated state is driven by IGF-1 and 2, IL-6, and the upregulation of the receptors PDGFR-beta and FGFR-1, 2, and 4. Deregulation of the cell cycle in this state involves reduction of p21 protein, concomitant upregulation of CDC2, and an increase in Rb phosphorylation that favors expression and nuclear translocation of FGFR-4 and FRAG-1 and 2. These events are associated with progressive activation of AKT and RAS. Malignant transformation is enhanced by near elimination of p21 and PC3, induction of AP-1 (upregulation of JUN-B, c-JUN, FRA-1), activation of the NF-kB pro-survival pathway, and inhibition of the TGF-beta pro-apoptotic pathway possibly in response to changes in the expression of nerve growth factor (NGF) I-A and NGFI-B. These data demonstrate that the events leading to malignancy in the rat brain in response to MNU treatment are to a great extent similar to those described for secondary glial malignancies in humans.

Methionine deprivation imposes a metabolic stress, termed methionine stress, that inhibits mitosis and induces cell cycle arrest and apoptosis. The methionine-dependent central nervous system tumor cell lines DAOY (medulloblastoma), SWB61 (anaplastic oligodendroglioma), SWB40 (anaplastic astrocytoma), and SWB39 (glioblastoma multiforme) were compared with methionine-stress resistant SWB77 (glioblastoma multiforme). The cDNA-oligoarray analysis and reverse transcription-PCR verification indicated common changes in gene expression in methionine-dependent cell lines to include up-regulation/induction of cyclin D1, mitotic arrest deficient (MAD)1, p21, growth arrest and DNA-damage-inducible (GADD)45 alpha, GADD45 gamma, GADD34, breast cancer (BRCA)1, 14-3-3sigma, B-cell CLL/lymphoma (BCL)1, transforming growth factor (TGF)-beta, TGF-beta-induced early response (TIEG), SMAD5, SMAD7, SMAD2, insulin-like growth factor binding protein (IGFBP7), IGF-R2, vascular endothelial growth factor (VEGF), TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha converting enzyme (TACE), TRAIL receptor (TRAIL-R)2, TNFR-related death receptor (DR)6, TRAF interacting protein (I-TRAF), IL-6, MDA7, IL-1B convertase (ICE)-gamma, delta and epsilon, IRF1, IRF5, IRF7, interferon (IFN)-gamma and receptor components, ISG15, p65-NF-kappaB, JUN-B, positive cofactor (PC)4, C/ERB-beta, inositol triphosphate receptor I, and methionine adenosyltransferase II. On the other hand, cyclins A1, A2, B1 and B2, cell division cycle (CDC)2 and its kinase, CDC25 A and B, budding uninhibited by benzimidazoles (BUB)1 and 3, MAD2, CDC28 protein kinase (CKS)1 and 2, neuroepithelial cell transforming gene (NET)1, activator of S-phase kinase (ASK), CDC14B phosphatase, BCL2, TGF-beta activated kinase (TAK)1, TAB1, c-FOS, DNA topoisomerase II, DNA polymerase alpha, dihydrofolate reductase, thymidine kinase, stathmin, and MAP4 were down-regulated. In the methionine stress-resistant SWB77, only 20% of the above genes were affected, and then only to a lesser extent. In addition, some of the changes observed in SWB77 were opposite to those seen in methionine-dependent tumors, including expression of p21, TRAIL-R2, and TIEG. Despite similarities, differences between methionine-dependent tumors were substantial, especially in regard to regulation of cytokine expression. Western blot analysis confirmed that methionine stress caused the following: (a) a marked increase of GADD45alpha and gamma in the wt-p53 cell lines SWB61 and 40; (b) an increase in GADD34 and p21 protein in all of the methionine-dependent lines; and (c) the induction of MDA7 and phospho-p38 in DAOY and SWB39, consistent with marked transcriptional activation of the former under methionine stress. It was additionally shown that methionine stress down-regulated the highly active phosphatidylinositol 3'-kinase pathway by reducing AKT phosphorylation, especially in DAOY and SWB77, and also reduced the levels of retinoblastoma (Rb) and pRb (P-ser780, P-ser795, and P-ser807/811), resulting in a shift in favor of unphosphorylated species in all of the methionine-dependent lines. Immunohistochemical analysis showed marked inhibition of nuclear translocation of nuclear factor kappaB under methionine stress in methionine-dependent lines. In this study we show for the first time that methionine stress mobilizes several defined cell cycle checkpoints and proapoptotic pathways while coordinately inhibiting prosurvival mechanisms in central nervous system tumors. It is clear that methionine stress-induced cytotoxicity is not restricted by the p53 mutational status.

The intracellular metabolites of long-term in vitro cultured axenic amastigotes of Leishmania donovani (strain Dd8) were determined and compared with those of promastigotes and intracellular amastigotes, employing proton NMR spectroscopy. The presence of two new metabolites, i.e. betaine and beta-hydroxybutyrate were reported. Betaine was detected in all the three stages being highest in the promastigotes while beta-hydroxybutyrate could be detected only in promastigotes and axenic amastigotes. Among other metabolites, succinate and valine were found in higher quantities in intracellular amastigotes and axenic amastigotes than in promastigotes. Acetoacetate was present only in axenic and intracellular amastigotes. The comparative metabolite profile of different parasite forms reveals that axenic amastigotes seem to represent an intermediate stage between promastigotes and intracellular amastigotes in spite of their strong resemblance to intracellular amastigotes in morphology, infectivity, biochemical studies and even in the manifestation of amastigote specific A2 protein.

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, alpha-glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.

In vitro as well as in vivo studies suggest that cytokine-induced synthesis of nitric oxide (NO) from L-arginine is a major effector mechanism against intracellular pathogens. In this study, we demonstrate that golden hamsters infected with Leishmania donovani amastigotes upon treatment with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), a potent interferon inducer and immune enhancer, in combination with L-arginine, develop the capacity to eliminate intracellular pathogens. This antileishmanial activity of poly ICLC was suppressed by N w nitro-L-arginine (N w NLA), an inhibitor of inducible NO synthase. Furthermore, prolonged treatment of infected animals with L-arginine alone for 5 days more after 5 day treatment with poly ICLC plus L-arginine increased the antileishmanial activity compared with 5 day treatment with poly ICLC plus L-arginine, suggesting that inducible NO synthase, once activated, produces NO for 5 days more. Our results suggest that an L-arginine-dependent, NO-mediated mechanism is probably responsible for the antileishmanial action of poly ICLC.

After presenting processed glycoprotein of Leishmania donovani to T-cell, macrophage seeks the help of a panel of T-cells lymphokines to transform from a state that sustains intra cellular replication of parasite to an effector state for destructing parasites. But esterase and trypsin of macrophage membrane prevent T-cells to release MIF. Role of soya-bean trypsin inhibitor (STI) has been exposed in the present study with a view to alter esterase functional behaviour of macrophage for control of T-cell activation and also, if T-cells once made responsive to antigen by STI do alter macrophage response to T-cells or not. Results establish STI as potent effector molecule, which can serve as an adjuvant to candidate T-cell epitope and synthetic peptide for development of anti-Kala-azar vaccine protocol in future.

The efficacy of sodium stibogluconate against Leishmania donovani infections was markedly enhanced by encapsulating this drug in tuftsin-bearing liposomes. Also, pretreatment of the animals with these liposomes (free of drug) rendered them resistant to this infection, possibly by activating the host's macrophages. These results demonstrate that tuftsin-bearing liposomes besides delivering the drug to the target cells could also enhance the nonspecific resistance against infections, thus offering an additional advantage over the use of tuftsin-free liposomes as drug carriers in leishmania therapy.