Parasitol Res. 2010 Feb 23. [Epub ahead of print]
Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis
Liu MM, Yuan ZG, Peng GH, Zhou DH, He XH, Yan C, Yin CC, He Y, Lin RQ, Song HQ, Zhu XQ.
Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province, 510642, People's Republic of China.
Microneme protein 8 (MIC8) is considered a new essential invasion factor in Toxoplasma gondii. In the present study, a deoxyribonucleic acid vaccine expressing MIC8 of T. gondii was constructed and the immune response it induced in Kunming mice was evaluated. The gene sequence encoding MIC8 was inserted into the eukaryotic expression vector pVAX I, and the pVAX-MIC8 expression plasmid was constructed, and the plasmid diluted with PBS to l00 mg/100 microl was injected into the Kunming mice muscularly. Levels of IgG antibody, gamma-interferon (IFN-gamma), interleukin-2 (IL-2), interleukin-4, and interleukin-10 were detected. The mice were challenged with tachyzoites of the virulent T. gondii RH strain at the 14th day after the last immunization to observe the survival time. The high level of IFN-gamma, IL-2, and IgG antibody indicated that mice vaccinated with recombinant pVAX-MIC8 plasmid could elicit strong cellular and humoral immune responses and showed a significantly increased survival time (10.3 +/- 0.9 days) compared with control mice which died within 5 days of challenge infection. These data demonstrate that the T. gondii MIC8 is a potential vaccine candidate against toxoplasmosis.
PMID: 20177910 [PubMed - as supplied by publisher]
Thursday, February 25, 2010
Tuesday, February 23, 2010
Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step
Eukaryot Cell. 2010 Feb 19. [Epub ahead of print]
Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step
Sugi T, Kato K, Kobayashi K, Watanabe S, Kurokawa H, Gong H, Pandey K, Takemae H, Akashi H.
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound, 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.
PMID: 20173034 [PubMed - as supplied by publisher]
Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step
Sugi T, Kato K, Kobayashi K, Watanabe S, Kurokawa H, Gong H, Pandey K, Takemae H, Akashi H.
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound, 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.
PMID: 20173034 [PubMed - as supplied by publisher]
Monday, February 22, 2010
Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells
Am J Pathol. 2010 Feb 18. [Epub ahead of print]
Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells
Suzuki Y, Wang X, Jortner BS, Payne L, Ni Y, Michie SA, Xu B, Kudo T, Perkins S.
From the Department of Biomedical Sciences,* Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia; the Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky; and the Department of Pathology, Stanford University School of Medicine, Stanford, California.
Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8(+) immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8(+) T cells does not require the production of interferon-gamma, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8(+) T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.
PMID: 20167872 [PubMed - as supplied by publisher]
Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells
Suzuki Y, Wang X, Jortner BS, Payne L, Ni Y, Michie SA, Xu B, Kudo T, Perkins S.
From the Department of Biomedical Sciences,* Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia; the Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky; and the Department of Pathology, Stanford University School of Medicine, Stanford, California.
Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8(+) immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8(+) T cells does not require the production of interferon-gamma, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8(+) T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.
PMID: 20167872 [PubMed - as supplied by publisher]
Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis
Auton Neurosci. 2010 Feb 17. [Epub ahead of print]
Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis
Odorizzi L, Moreira NM, Gonçalves GF, da Silva AV, Sant'ana DD, Araújo EJ.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, PR, Brazil.
The consequences of the infection caused by Toxoplasma gondii in myenteric neurons of the jejunum of swines reactive to NADH-diaphorase and NADPH-diaphorase were evaluated in this study. Ten 88-day-old mixed-breed swines (Pietrain and Wessex) were assigned into two groups: Control (n=5) and Experimental (n=5), which orally received 5000 sporulated oocysts from a genotype III T. gondii strain. After 30days, the animals were anesthetized, having part of their jejunum removed and stained with NADPH-diaphorase and NADH-diaphorase. NADPHd-p neurons (nitrergic) presented increase of the number of cells per ganglion and hypertrophy. The number of NADHd-p neurons (metabolic more active) and their nuclear area decreased. Copyright © 2010. Published by Elsevier B.V.
PMID: 20167543 [PubMed - as supplied by publisher]
Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis
Odorizzi L, Moreira NM, Gonçalves GF, da Silva AV, Sant'ana DD, Araújo EJ.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, PR, Brazil.
The consequences of the infection caused by Toxoplasma gondii in myenteric neurons of the jejunum of swines reactive to NADH-diaphorase and NADPH-diaphorase were evaluated in this study. Ten 88-day-old mixed-breed swines (Pietrain and Wessex) were assigned into two groups: Control (n=5) and Experimental (n=5), which orally received 5000 sporulated oocysts from a genotype III T. gondii strain. After 30days, the animals were anesthetized, having part of their jejunum removed and stained with NADPH-diaphorase and NADH-diaphorase. NADPHd-p neurons (nitrergic) presented increase of the number of cells per ganglion and hypertrophy. The number of NADHd-p neurons (metabolic more active) and their nuclear area decreased. Copyright © 2010. Published by Elsevier B.V.
PMID: 20167543 [PubMed - as supplied by publisher]
Saturday, February 20, 2010
Structural and functional characterization of SporoSAG: A SAG2 related surface antigen from Toxoplasma
J Biol Chem. 2010 Feb 17. [Epub ahead of print]
Structural and functional characterization of SporoSAG: A SAG2 related surface antigen from Toxoplasma gondii
Crawford J, Lamb E, Wasmuth J, Grujic O, Grigg ME, Boulanger MJ.
University of Victoria, Canada;
Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG (Surface Antigen Glycoprotein) clusters with the SAG2 subfamily of the SAG1 related superfamily (SRS) and is nonpolymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 A resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS beta sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the beta sandwich fold and may have important implications for dimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.
PMID: 20164173 [PubMed - as supplied by publisher]
Structural and functional characterization of SporoSAG: A SAG2 related surface antigen from Toxoplasma gondii
Crawford J, Lamb E, Wasmuth J, Grujic O, Grigg ME, Boulanger MJ.
University of Victoria, Canada;
Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to large scale outbreaks of toxoplasmosis. SporoSAG is the dominant surface coat protein expressed on the surface of sporozoites. Using a bioinformatic approach, we show that SporoSAG (Surface Antigen Glycoprotein) clusters with the SAG2 subfamily of the SAG1 related superfamily (SRS) and is nonpolymorphic among the 11 haplogroups of T. gondii strains. In contrast to the immunodominant SAG1 protein expressed on tachyzoites, SporoSAG is non-immunogenic during natural infection. We report the 1.60 A resolution crystal structure of SporoSAG solved using cadmium single anomalous dispersion. SporoSAG crystallized as a monomer and displays unique features of the SRS beta sandwich fold relative to SAG1 and BSR4. Intriguingly, the structural diversity is localized to the upper sheets of the beta sandwich fold and may have important implications for dimerization and host cell ligand recognition. The structure of SporoSAG also reveals an unexpectedly acidic surface that contrasts with the previously determined SAG1 and BSR4 structures where a basic surface is predicted to play a role in binding negatively charged glycosaminoglycans. Our structural and functional characterization of SporoSAG provides a rationale for the evolutionary divergence of this key SRS family member.
PMID: 20164173 [PubMed - as supplied by publisher]
Thursday, February 18, 2010
MYST-family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated DNA damage response in Toxoplasma
J Biol Chem. 2010 Feb 16. [Epub ahead of print]
MYST-family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated DNA damage response in Toxoplasma gondii
Vonlaufen N, Naguleswaran A, Coppens I, Sullivan WJ Jr.
Indiana University School of Medicine, United States;
The MYST family of lysine acetyltransferases (KATs) function in a wide variety of cellular operations, including gene regulation and the DNA damage response. Here we report the characterization of the second MYST family KAT in the protozoan parasite Toxoplasma gondii (TgMYST-B). Toxoplasma causes birth defects and is an opportunistic pathogen in the immunocompromised, the latter due to its ability to convert into a latent cyst (bradyzoite). We demonstrate that TgMYST-B can gain access to the parasite nucleus and acetylate histones. Over-expression of recombinant, tagged TgMYST-B reduces growth rate in vitro and confers protection from a DNA alkylating agent. Expression of mutant TgMYST-B produced no growth defect and failed to protect against DNA damage. We demonstrate that cells over-expressing TgMYST-B have increased levels of ATM kinase and phosphorylated H2AX, and that TgMYST-B localizes to the ATM kinase gene. Pharmacological inhibitors of ATM kinase or KATs reverse the slow growth phenotype seen in parasites over-expressing TgMYST-B. These studies are the first to show that a MYST KAT contributes to ATM kinase gene expression, further illuminating the mechanism of how ATM kinase is upregulated to respond to DNA damage.
PMID: 20159970 [PubMed - as supplied by publisher]
MYST-family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated DNA damage response in Toxoplasma gondii
Vonlaufen N, Naguleswaran A, Coppens I, Sullivan WJ Jr.
Indiana University School of Medicine, United States;
The MYST family of lysine acetyltransferases (KATs) function in a wide variety of cellular operations, including gene regulation and the DNA damage response. Here we report the characterization of the second MYST family KAT in the protozoan parasite Toxoplasma gondii (TgMYST-B). Toxoplasma causes birth defects and is an opportunistic pathogen in the immunocompromised, the latter due to its ability to convert into a latent cyst (bradyzoite). We demonstrate that TgMYST-B can gain access to the parasite nucleus and acetylate histones. Over-expression of recombinant, tagged TgMYST-B reduces growth rate in vitro and confers protection from a DNA alkylating agent. Expression of mutant TgMYST-B produced no growth defect and failed to protect against DNA damage. We demonstrate that cells over-expressing TgMYST-B have increased levels of ATM kinase and phosphorylated H2AX, and that TgMYST-B localizes to the ATM kinase gene. Pharmacological inhibitors of ATM kinase or KATs reverse the slow growth phenotype seen in parasites over-expressing TgMYST-B. These studies are the first to show that a MYST KAT contributes to ATM kinase gene expression, further illuminating the mechanism of how ATM kinase is upregulated to respond to DNA damage.
PMID: 20159970 [PubMed - as supplied by publisher]
The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis
J Ocul Biol Dis Infor. 2009 Dec 12;2(4):214-222.
The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis
Brown KM, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73034 USA.
Ocular toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, is the leading cause of retinochoroiditis. Toxoplasma is an obligate intracellular pathogen that replicates within a parasitophorous vacuole. Infections are initiated by digestion of parasites deposited in cat feces or in undercooked meat. Parasites then disseminate to target tissues that include the retina where they then develop into long-lived asymptomatic tissue cysts. Occasionally, cysts reactivate and growth of newly emerged parasites must be controlled by the host's immune system or disease will occur. The mechanisms by which Toxoplasma grows within its host cell, encysts, and interacts with the host's immune system are important questions. Here, we will discuss how the use of DNA microarrays in transcriptional profiling, genotyping, and epigenetic experiments has impacted our understanding of these processes. Finally, we will discuss how these advances relate to ocular toxoplasmosis and how future research on ocular toxoplasmosis can benefit from DNA microarrays.
PMID: 20157353 [PubMed]
The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis
Brown KM, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73034 USA.
Ocular toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, is the leading cause of retinochoroiditis. Toxoplasma is an obligate intracellular pathogen that replicates within a parasitophorous vacuole. Infections are initiated by digestion of parasites deposited in cat feces or in undercooked meat. Parasites then disseminate to target tissues that include the retina where they then develop into long-lived asymptomatic tissue cysts. Occasionally, cysts reactivate and growth of newly emerged parasites must be controlled by the host's immune system or disease will occur. The mechanisms by which Toxoplasma grows within its host cell, encysts, and interacts with the host's immune system are important questions. Here, we will discuss how the use of DNA microarrays in transcriptional profiling, genotyping, and epigenetic experiments has impacted our understanding of these processes. Finally, we will discuss how these advances relate to ocular toxoplasmosis and how future research on ocular toxoplasmosis can benefit from DNA microarrays.
PMID: 20157353 [PubMed]
NF-kappaB1 contributes to T cell-mediated control of Toxoplasma gondii in the CNS
J Neuroimmunol. 2010 Feb 13. [Epub ahead of print]
NF-kappaB1 contributes to T cell-mediated control of Toxoplasma gondii in the CNS
Harris TH, Wilson EH, Tait ED, Buckley M, Shapira S, Caamano J, Artis D, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 S University Ave, Philadelphia, PA 19104, United States.
In this study, the role of NF-kappaB1 was examined during toxoplasmosis. While wildtype BALB/c mice generated protective responses, NF-kappaB1(-/-) mice developed Toxoplasmic encephalitis, characterized by increased parasite burden and necrosis in the brain. Susceptibility was primarily associated with a local decrease in the number of CD8(+) T cells and IFN-gamma production, while accessory cell function appeared intact in NF-kappaB1(-/-) mice. Consistent with these findings, T cell transfer studies revealed that NF-kappaB1(-/-) T cells provided SCID mice less protection than wildtype T cells. These results demonstrate an intrinsic role for NF-kappaB1 in T cell-mediated immunity to Toxoplasmagondii. Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 20156658 [PubMed - as supplied by publisher]
NF-kappaB1 contributes to T cell-mediated control of Toxoplasma gondii in the CNS
Harris TH, Wilson EH, Tait ED, Buckley M, Shapira S, Caamano J, Artis D, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 S University Ave, Philadelphia, PA 19104, United States.
In this study, the role of NF-kappaB1 was examined during toxoplasmosis. While wildtype BALB/c mice generated protective responses, NF-kappaB1(-/-) mice developed Toxoplasmic encephalitis, characterized by increased parasite burden and necrosis in the brain. Susceptibility was primarily associated with a local decrease in the number of CD8(+) T cells and IFN-gamma production, while accessory cell function appeared intact in NF-kappaB1(-/-) mice. Consistent with these findings, T cell transfer studies revealed that NF-kappaB1(-/-) T cells provided SCID mice less protection than wildtype T cells. These results demonstrate an intrinsic role for NF-kappaB1 in T cell-mediated immunity to Toxoplasmagondii. Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 20156658 [PubMed - as supplied by publisher]
Tuesday, February 16, 2010
Characterization of alpha-phosphoglucomutase isozymes from Toxoplasma
Parasitol Int. 2010 Feb 11. [Epub ahead of print]
Characterization of alpha-phosphoglucomutase isozymes from Toxoplasma gondii
Imada M, Kawashima S, Kanehisa M, Takeuchi T, Asai T.
Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
The Toxoplasma gondii genome project has revealed two putative isoforms (TgPGM-I and TgPGM-II) of alpha-phosphoglucomutase (EC 5.4.2.2). We obtained recombinant proteins of these isoforms from the Beverley strain of T. gondii and characterized their properties, particularly the kinetic properties of these isoforms. The specific activities of TgPGM-I and TgPGM-II foralpha-D-glucose 1-phosphate were 338+/-9 and 84+/-6mumol/min/mg protein, respectively, at 37 degrees C under optimal conditions. The Kcat and Km values of TgPGM-I were 398 +/- 11 /s and 0.19+/-0.03mM and those for TgPGM-II were 93+/-7 /s and 3.53+/-0.91mM, respectively, foralpha-D-glucose 1-phosphate. Magnesium ions were the most effective divalent cations for both the enzyme activities. The maximum activities of both the enzymes were obtained in the presence of more than 0.2 mMalpha-D-glucose 1,6-bisphosphate. Although both enzymes were attached to thealpha-phosphohexomutase superfamily, amino acid sequence homology between TgPGM-I and TgPGM-II showed very low overall identity (25%). Noalpha-phosphomannomutase (EC 5.4.2.8) activity was detected for either enzyme. The data indicated that TgPGM-I, but not TgPGM-II, may play an important role in alpha-D-glucose 6-phosphate production. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.
PMID: 20153838 [PubMed - as supplied by publisher]
Characterization of alpha-phosphoglucomutase isozymes from Toxoplasma gondii
Imada M, Kawashima S, Kanehisa M, Takeuchi T, Asai T.
Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
The Toxoplasma gondii genome project has revealed two putative isoforms (TgPGM-I and TgPGM-II) of alpha-phosphoglucomutase (EC 5.4.2.2). We obtained recombinant proteins of these isoforms from the Beverley strain of T. gondii and characterized their properties, particularly the kinetic properties of these isoforms. The specific activities of TgPGM-I and TgPGM-II foralpha-D-glucose 1-phosphate were 338+/-9 and 84+/-6mumol/min/mg protein, respectively, at 37 degrees C under optimal conditions. The Kcat and Km values of TgPGM-I were 398 +/- 11 /s and 0.19+/-0.03mM and those for TgPGM-II were 93+/-7 /s and 3.53+/-0.91mM, respectively, foralpha-D-glucose 1-phosphate. Magnesium ions were the most effective divalent cations for both the enzyme activities. The maximum activities of both the enzymes were obtained in the presence of more than 0.2 mMalpha-D-glucose 1,6-bisphosphate. Although both enzymes were attached to thealpha-phosphohexomutase superfamily, amino acid sequence homology between TgPGM-I and TgPGM-II showed very low overall identity (25%). Noalpha-phosphomannomutase (EC 5.4.2.8) activity was detected for either enzyme. The data indicated that TgPGM-I, but not TgPGM-II, may play an important role in alpha-D-glucose 6-phosphate production. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.
PMID: 20153838 [PubMed - as supplied by publisher]
Thursday, February 11, 2010
Dinitroaniline Activity in Toxoplasma gondii Expressing Wild-type or Mutant Alpha-Tubulin
Antimicrob Agents Chemother. 2010 Feb 9. [Epub ahead of print]
Dinitroaniline Activity in Toxoplasma gondii Expressing Wild-type or Mutant Alpha-Tubulin
Ma C, Tran J, Gu F, Ochoa R, Li C, Sept D, Werbovetz K, Morrissette N.
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, 92697; Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109-2099; Division of Medicinal Chemistry & Pharmacognosy, Ohio State University, 332 Parks Hall, 500 West 12th Avenue, Columbus, OH 43210-1291; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
The human parasite Toxoplasma gondii is sensitive to dinitroaniline compounds which selectively disrupt microtubules in diverse protozoa but which have no detectable effect on vertebrate host cell microtubules or other functions. Replication of wild type T. gondii is inhibited by 0.5-2.5 muM oryzalin, but mutant parasites harboring amino acid substitutions in the predicted dinitroaniline binding site confer resistance up to 40 muM oryzalin. However, the precise interaction between dinitroanilines and the binding site in alpha-tubulin remains unclear. We have investigated the activity of 12 dinitroanilines and the related compound amiprophos methyl on wild-type and dinitroaniline-resistant parasite lines that contain proposed binding site mutations. These data indicate that dinitramine is the most effective dinitroaniline to inhibit Toxoplasma growth in wild-type parasites and most resistant lines. Dinitramine has an amine group at the meta position not present in any of the other dinitroanilines tested here that is predicted to form hydrogen-bonds with residues Arg2 and Gln133 according to docking data. Remarkably, although the binding site mutation Ile235Val confers increased resistance to most dinitroanilines, it confers increased sensitivity to GB-II-5, a compound optimized for activity against kinetoplastid tubulin. Kinetoplastid parasites have a valine at position 235 of alpha-tubulin whereas apicomplexan parasites have an isoleucine at this site. We suggest that this heterogeneity in binding site environment influences relative dinitroaniline sensitivity in distinct protozoan lineages and hypothesize that a mutation that makes the apicomplexan dinitroaniline binding site more like the kinetoplastid site increases sensitivity to a dinitroaniline optimized for activity in the latter parasites.
PMID: 20145086 [PubMed - as supplied by publisher]
Dinitroaniline Activity in Toxoplasma gondii Expressing Wild-type or Mutant Alpha-Tubulin
Ma C, Tran J, Gu F, Ochoa R, Li C, Sept D, Werbovetz K, Morrissette N.
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, 92697; Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109-2099; Division of Medicinal Chemistry & Pharmacognosy, Ohio State University, 332 Parks Hall, 500 West 12th Avenue, Columbus, OH 43210-1291; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
The human parasite Toxoplasma gondii is sensitive to dinitroaniline compounds which selectively disrupt microtubules in diverse protozoa but which have no detectable effect on vertebrate host cell microtubules or other functions. Replication of wild type T. gondii is inhibited by 0.5-2.5 muM oryzalin, but mutant parasites harboring amino acid substitutions in the predicted dinitroaniline binding site confer resistance up to 40 muM oryzalin. However, the precise interaction between dinitroanilines and the binding site in alpha-tubulin remains unclear. We have investigated the activity of 12 dinitroanilines and the related compound amiprophos methyl on wild-type and dinitroaniline-resistant parasite lines that contain proposed binding site mutations. These data indicate that dinitramine is the most effective dinitroaniline to inhibit Toxoplasma growth in wild-type parasites and most resistant lines. Dinitramine has an amine group at the meta position not present in any of the other dinitroanilines tested here that is predicted to form hydrogen-bonds with residues Arg2 and Gln133 according to docking data. Remarkably, although the binding site mutation Ile235Val confers increased resistance to most dinitroanilines, it confers increased sensitivity to GB-II-5, a compound optimized for activity against kinetoplastid tubulin. Kinetoplastid parasites have a valine at position 235 of alpha-tubulin whereas apicomplexan parasites have an isoleucine at this site. We suggest that this heterogeneity in binding site environment influences relative dinitroaniline sensitivity in distinct protozoan lineages and hypothesize that a mutation that makes the apicomplexan dinitroaniline binding site more like the kinetoplastid site increases sensitivity to a dinitroaniline optimized for activity in the latter parasites.
PMID: 20145086 [PubMed - as supplied by publisher]
Characterization of a novel thrombospondin-related protein in Toxoplasma
Parasitol Int. 2010 Feb 5. [Epub ahead of print]
Characterization of a novel thrombospondin-related protein in Toxoplasma gondii
Kawase O, Nishikawa Y, Bannai H, Igarashi M, Matsuo T, Xuan X.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades a wide range of host cells. The parasite releases a large variety of proteins from a secretory organelle, microneme, and the secretion is essential for the parasite invasion. We cloned a secreted protein with an altered thrombospondin repeat of Toxoplasma gondii (TgSPATR), which was the homologue of Plasmodium SPATRs. Immunofluorescence double staining experiment revealed that TgSPATR was co-localized with a microneme protein, MIC2, and immuno-electron microscopic (IEM) analysis detected TgSPATR in the microneme-like structure. TgSPATR secretion was induced by ethanol, while an intracellular Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), suppressed the ethanol-induced secretion, suggesting the secretion was Ca(2+)-dependent, similarly to known microneme proteins. Furthermore, TgSPATR, existed on outer surface of the parasites, was detected by incomplete membrane permeabilization by saponin and immunofluorescent antibody test (IFAT). Both TgSPATR and MIC2 were detected on outer surface of extracellular parasites, but not of intracellular single parasites, suggesting they were similarly secreted during early stages of parasite invasion. Therefore, TgSPATR is probably new member of microneme protein and maybe involved in parasite invasion. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.
PMID: 20144733 [PubMed - as supplied by publisher]
Characterization of a novel thrombospondin-related protein in Toxoplasma gondii
Kawase O, Nishikawa Y, Bannai H, Igarashi M, Matsuo T, Xuan X.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades a wide range of host cells. The parasite releases a large variety of proteins from a secretory organelle, microneme, and the secretion is essential for the parasite invasion. We cloned a secreted protein with an altered thrombospondin repeat of Toxoplasma gondii (TgSPATR), which was the homologue of Plasmodium SPATRs. Immunofluorescence double staining experiment revealed that TgSPATR was co-localized with a microneme protein, MIC2, and immuno-electron microscopic (IEM) analysis detected TgSPATR in the microneme-like structure. TgSPATR secretion was induced by ethanol, while an intracellular Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), suppressed the ethanol-induced secretion, suggesting the secretion was Ca(2+)-dependent, similarly to known microneme proteins. Furthermore, TgSPATR, existed on outer surface of the parasites, was detected by incomplete membrane permeabilization by saponin and immunofluorescent antibody test (IFAT). Both TgSPATR and MIC2 were detected on outer surface of extracellular parasites, but not of intracellular single parasites, suggesting they were similarly secreted during early stages of parasite invasion. Therefore, TgSPATR is probably new member of microneme protein and maybe involved in parasite invasion. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.
PMID: 20144733 [PubMed - as supplied by publisher]
Inflammatory Monocytes but not Neutrophils are Necessary to Control Infection with Toxoplasma gondii in Mice
Infect Immun. 2010 Feb 9. [Epub ahead of print]
Inflammatory Monocytes but not Neutrophils are Necessary to Control Infection with Toxoplasma gondii in Mice
Dunay IR, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110; Department of Neuropathology, University of Freiberg, Breisacherstrasse 64, Freiberg, Germany, 79106.
Previous studies have suggested that both inflammatory monocytes and neutrophils are important for controlling acute toxoplasmosis in the mouse model. To test the role of these cell types, we used mAb RB6-8C5 to deplete both subsets of cells, or mAb 1A8 to selectively remove neutrophils. RB6-8C5 mAb-treated mice succumbed to oral infection with T. gondii, similarly to Ccr2(-/-) mice, which are deficient in monocyte recruitment but have normal neutrophils. In contrast, mice treated with mAb 1A8 controlled parasite replication and survived acute infection. Ccr2(-/-) mice suffered from acute ileitis and inflammation in the spleen that was associated with a lack of inflammatory monocytes and elevated numbers of neutrophils. RB6-8C5 mAb-treated C57BL/6 mice also suffered from intestinal pathology and splenic damage, although this was less extensive due to reduced numbers of neutrophils. Neutrophil-depleted infected wild type mice displayed no pathological changes, compared to untreated infected controls. Collectively, these observations demonstrate the critical role of inflammatory monocytes during the acute infection with the parasite T. gondii, and reveal that neutrophils are not protective but rather contribute to the pathology.
PMID: 20145099 [PubMed - as supplied by publisher]
Inflammatory Monocytes but not Neutrophils are Necessary to Control Infection with Toxoplasma gondii in Mice
Dunay IR, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110; Department of Neuropathology, University of Freiberg, Breisacherstrasse 64, Freiberg, Germany, 79106.
Previous studies have suggested that both inflammatory monocytes and neutrophils are important for controlling acute toxoplasmosis in the mouse model. To test the role of these cell types, we used mAb RB6-8C5 to deplete both subsets of cells, or mAb 1A8 to selectively remove neutrophils. RB6-8C5 mAb-treated mice succumbed to oral infection with T. gondii, similarly to Ccr2(-/-) mice, which are deficient in monocyte recruitment but have normal neutrophils. In contrast, mice treated with mAb 1A8 controlled parasite replication and survived acute infection. Ccr2(-/-) mice suffered from acute ileitis and inflammation in the spleen that was associated with a lack of inflammatory monocytes and elevated numbers of neutrophils. RB6-8C5 mAb-treated C57BL/6 mice also suffered from intestinal pathology and splenic damage, although this was less extensive due to reduced numbers of neutrophils. Neutrophil-depleted infected wild type mice displayed no pathological changes, compared to untreated infected controls. Collectively, these observations demonstrate the critical role of inflammatory monocytes during the acute infection with the parasite T. gondii, and reveal that neutrophils are not protective but rather contribute to the pathology.
PMID: 20145099 [PubMed - as supplied by publisher]
Tuesday, February 09, 2010
TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii
PLoS Pathog. 2010 Feb 5;6(2):e1000754.
TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii
Heaslip AT, Dzierszinski F, Stein B, Hu K.
Department of Biology, Indiana University, Bloomington, Indiana, United States of America.
Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (DeltaTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in DeltaTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, DeltaTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.
PMID: 20140195 [PubMed - as supplied by publisher]
TgMORN1 Is a Key Organizer for the Basal Complex of Toxoplasma gondii
Heaslip AT, Dzierszinski F, Stein B, Hu K.
Department of Biology, Indiana University, Bloomington, Indiana, United States of America.
Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (DeltaTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in DeltaTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, DeltaTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.
PMID: 20140195 [PubMed - as supplied by publisher]
Monday, February 08, 2010
The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18
Res Vet Sci. 2010 Feb 2. [Epub ahead of print]
The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18
Liu Q, Shang L, Jin H, Wei F, Zhu XQ, Gao H.
Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130062, Jilin Province, China.
More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3weeks apart, and challenged with T. gondii 3weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-gamma and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P<0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii. Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 20132954 [PubMed - as supplied by publisher]
The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18
Liu Q, Shang L, Jin H, Wei F, Zhu XQ, Gao H.
Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130062, Jilin Province, China.
More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3weeks apart, and challenged with T. gondii 3weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-gamma and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P<0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii. Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 20132954 [PubMed - as supplied by publisher]
Parasiticidal activity of human alpha-defensin-5 against Toxoplasma
In Vitro Cell Dev Biol Anim. 2010 Feb 5. [Epub ahead of print]
Parasiticidal activity of human alpha-defensin-5 against Toxoplasma gondii
Tanaka T, Rahman MM, Battur B, Boldbaatar D, Liao M, Umemiya-Shirafuji R, Xuan X, Fujisaki K.
Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Science, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan.
Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. In this paper, we show that human alpha-defensin-5 displays a parasiticidal role against Toxoplasma gondii, the causative agent of toxoplasmosis. Exposure of the tachyzoite form of T. gondii to defensin induced aggregation and significantly reduced parasite viability in a concentration-dependent peptide. Pre-incubation of tachyzoites with human alpha-defensin-5 followed by exposure to a mouse embryonal cell line (NIH/3T3) significantly reduced T. gondii infection in these cells. Thus, human alpha-defensin-5 is an innate immune molecule that causes severe toxocity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that human alpha-defensin-5 causes aggregation, leading to Toxoplasma destruction.
PMID: 20135360 [PubMed - as supplied by publisher]
Parasiticidal activity of human alpha-defensin-5 against Toxoplasma gondii
Tanaka T, Rahman MM, Battur B, Boldbaatar D, Liao M, Umemiya-Shirafuji R, Xuan X, Fujisaki K.
Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Science, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan.
Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. In this paper, we show that human alpha-defensin-5 displays a parasiticidal role against Toxoplasma gondii, the causative agent of toxoplasmosis. Exposure of the tachyzoite form of T. gondii to defensin induced aggregation and significantly reduced parasite viability in a concentration-dependent peptide. Pre-incubation of tachyzoites with human alpha-defensin-5 followed by exposure to a mouse embryonal cell line (NIH/3T3) significantly reduced T. gondii infection in these cells. Thus, human alpha-defensin-5 is an innate immune molecule that causes severe toxocity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that human alpha-defensin-5 causes aggregation, leading to Toxoplasma destruction.
PMID: 20135360 [PubMed - as supplied by publisher]
Friday, February 05, 2010
The evolution, metabolism and functions of the apicoplast
Philos Trans R Soc Lond B Biol Sci. 2010 Mar 12;365(1541):749-63.
The evolution, metabolism and functions of the apicoplast
Lim L, McFadden GI.
School of Botany, University of Melbourne, , Parkville, Victoria 3010, Australia.
The malaria parasite, Plasmodium falciparum, harbours a relict plastid known as the 'apicoplast'. The discovery of the apicoplast ushered in an exciting new prospect for drug development against the parasite. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions. Morphological, biochemical and bioinformatic studies of the apicoplast have further reinforced its 'plant-like' characteristics and potential as a drug target. However, we are still not sure why the apicoplast is essential for the parasite's survival. This review explores the origins and metabolic functions of the apicoplast. In an attempt to decipher the role of the organelle within the parasite we also take a closer look at the transporters decorating the plastid to better understand the metabolic exchanges between the apicoplast and the rest of the parasite cell.
PMID: 20124342 [PubMed - in process]
The evolution, metabolism and functions of the apicoplast
Lim L, McFadden GI.
School of Botany, University of Melbourne, , Parkville, Victoria 3010, Australia.
The malaria parasite, Plasmodium falciparum, harbours a relict plastid known as the 'apicoplast'. The discovery of the apicoplast ushered in an exciting new prospect for drug development against the parasite. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions. Morphological, biochemical and bioinformatic studies of the apicoplast have further reinforced its 'plant-like' characteristics and potential as a drug target. However, we are still not sure why the apicoplast is essential for the parasite's survival. This review explores the origins and metabolic functions of the apicoplast. In an attempt to decipher the role of the organelle within the parasite we also take a closer look at the transporters decorating the plastid to better understand the metabolic exchanges between the apicoplast and the rest of the parasite cell.
PMID: 20124342 [PubMed - in process]
GRA1 in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages
Exp Parasitol. 2010 Jan 29. [Epub ahead of print]
Toxoplasma gondii: Expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages
Lin J, Lin X, Yang GH, Wang Y, Peng BW, Lin JY.
Department of Molecular Medicine, Fujian medical University, Fuzhou, Fujian, P.R.China.
In this study, effects of GRA1 organelle-targeted expression on macrophage functions were investigated. The recombinant plasmid pCMV/myc/ER-GRA1 was constructed and then was transfected into murine macrophage RAW264.7 by Lipofectamine, selected by resistance of G418. The selected mono-clone cell line was named ER-GRA1-RAW264.7. The expression of GRA1 was localized in ER of ER-GRA1-RAW264.7 cells by indirect immunofluorescence detection. GRA1 mRNA expression level in ER-GRA1-RAW264.7 cell was significantly enhanced with a concomitant increase in its growth and adherence activity. Fluorescence intensity of intracellular calcium in ER-GRA1-RAW264.7, ER-ctrl-RAW264.7 and RAW264.7 cells in the presence of 1 mmol/l Arachidonic Acid (AA) were assayed by confocal microscopy using calcium-sensitive dye, Fluo-3 AM. Cytoplasm [Ca2+]i peaked at about 18 s after AA treatment, and cytoplasm [Ca2+]i of RAW264.7 cell almost instantly stepped up after AA was added, and peaked in 3 s, with a minor cytoplasm [Ca2+]i vibration subsequently. These results demonstrated that the expression of GRA1 in ER of macrophages promotes both growth and adherence of macrophages and modulates the intracellular calcium release stimulated by AA. Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 20122928 [PubMed - as supplied by publisher]
Toxoplasma gondii: Expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages
Lin J, Lin X, Yang GH, Wang Y, Peng BW, Lin JY.
Department of Molecular Medicine, Fujian medical University, Fuzhou, Fujian, P.R.China.
In this study, effects of GRA1 organelle-targeted expression on macrophage functions were investigated. The recombinant plasmid pCMV/myc/ER-GRA1 was constructed and then was transfected into murine macrophage RAW264.7 by Lipofectamine, selected by resistance of G418. The selected mono-clone cell line was named ER-GRA1-RAW264.7. The expression of GRA1 was localized in ER of ER-GRA1-RAW264.7 cells by indirect immunofluorescence detection. GRA1 mRNA expression level in ER-GRA1-RAW264.7 cell was significantly enhanced with a concomitant increase in its growth and adherence activity. Fluorescence intensity of intracellular calcium in ER-GRA1-RAW264.7, ER-ctrl-RAW264.7 and RAW264.7 cells in the presence of 1 mmol/l Arachidonic Acid (AA) were assayed by confocal microscopy using calcium-sensitive dye, Fluo-3 AM. Cytoplasm [Ca2+]i peaked at about 18 s after AA treatment, and cytoplasm [Ca2+]i of RAW264.7 cell almost instantly stepped up after AA was added, and peaked in 3 s, with a minor cytoplasm [Ca2+]i vibration subsequently. These results demonstrated that the expression of GRA1 in ER of macrophages promotes both growth and adherence of macrophages and modulates the intracellular calcium release stimulated by AA. Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 20122928 [PubMed - as supplied by publisher]
Tuesday, February 02, 2010
CoMFA analysis of tgDHFR and rlDHFR based on antifolates with 6-5 fused ring system
Bioorg Med Chem. 2010 Jan 6. [Epub ahead of print]
CoMFA analysis of tgDHFR and rlDHFR based on antifolates with 6-5 fused ring system using the all-orientation search (AOS) routine and a modified cross-validated r(2)-guided region selection (q(2)-GRS) routine and its initial application
Gangjee A, Lin X, Biondo LR, Queener SF.
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States.
We report the development of CoMFA analysis models that correlate the 3D chemical structures of 80 compounds with 6-5 fused ring system synthesized in our laboratory and their inhibitory potencies against tgDHFR and rlDHFR. In addition to conventional CoMFA analysis, we used two routines available in the literature aimed at the optimization of CoMFA: all-orientation search (AOS) and cross-validated r(2)-guided region selection (q(2)-GRS) to further optimize the models. During this process, we identified a problem associated with q(2)-GRS routine and modified using two strategies. Thus, for the inhibitory activity against each enzyme (tgDHFR and rlDHFR), five CoMFA models were developed using the conventional CoMFA, AOS optimized CoMFA, the original q(2)-GRS optimized CoMFA and the modified q(2)-GRS optimized CoMFA using the first and the second strategy. In this study, we demonstrate that the modified q(2)-GRS routines are superior to the original routine. On the basis of the steric contour maps of the models, we designed four new compounds in the 2,4-diamino-5-methyl-6-phenylsulfanyl-substituted pyrrolo[2,3-d]pyrimidine series. As predicted, the new compounds were potent and selective inhibitors of tgDHFR. One of them, 2,4-diamino-5-methyl-6-(2',6'-dimethylphenylthio)pyrrolo[2,3-d]pyrimidine, is the first 6-5 fused ring system compound with nanomolar tgDHFR inhibitory activity. The HCl salt of this compound was also prepared to increase solubility. Both forms of the drug were tested in vivo in a Toxoplasma gondii infection mouse model. The results indicate that both forms were active with the HCl salt significantly more potent than the free base. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20117005 [PubMed - as supplied by publisher]
CoMFA analysis of tgDHFR and rlDHFR based on antifolates with 6-5 fused ring system using the all-orientation search (AOS) routine and a modified cross-validated r(2)-guided region selection (q(2)-GRS) routine and its initial application
Gangjee A, Lin X, Biondo LR, Queener SF.
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States.
We report the development of CoMFA analysis models that correlate the 3D chemical structures of 80 compounds with 6-5 fused ring system synthesized in our laboratory and their inhibitory potencies against tgDHFR and rlDHFR. In addition to conventional CoMFA analysis, we used two routines available in the literature aimed at the optimization of CoMFA: all-orientation search (AOS) and cross-validated r(2)-guided region selection (q(2)-GRS) to further optimize the models. During this process, we identified a problem associated with q(2)-GRS routine and modified using two strategies. Thus, for the inhibitory activity against each enzyme (tgDHFR and rlDHFR), five CoMFA models were developed using the conventional CoMFA, AOS optimized CoMFA, the original q(2)-GRS optimized CoMFA and the modified q(2)-GRS optimized CoMFA using the first and the second strategy. In this study, we demonstrate that the modified q(2)-GRS routines are superior to the original routine. On the basis of the steric contour maps of the models, we designed four new compounds in the 2,4-diamino-5-methyl-6-phenylsulfanyl-substituted pyrrolo[2,3-d]pyrimidine series. As predicted, the new compounds were potent and selective inhibitors of tgDHFR. One of them, 2,4-diamino-5-methyl-6-(2',6'-dimethylphenylthio)pyrrolo[2,3-d]pyrimidine, is the first 6-5 fused ring system compound with nanomolar tgDHFR inhibitory activity. The HCl salt of this compound was also prepared to increase solubility. Both forms of the drug were tested in vivo in a Toxoplasma gondii infection mouse model. The results indicate that both forms were active with the HCl salt significantly more potent than the free base. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20117005 [PubMed - as supplied by publisher]
Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii
Exp Parasitol. 2010 Jan 28. [Epub ahead of print]
Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii
Sukhumavasi W, Warren AL, Alonso LD, Denkers EY.
Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
The function of mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK)-2 in resistance and pathology during infection has not been greatly studied. Here, we employed Jnk2(-/-) mice to investigate the role of JNK2 in resistance and immunity during oral infection with the protozoan pathogen Toxoplasma gondii. We found increased host resistance in the absence of JNK2 as determined by lower parasite burden and increased host survival. Lack of JNK2 also correlated with decreased neutrophil recruitment to the intestinal mucosa and less pathology in the small intestine. In the absence of JNK2, IL-12 production was slightly but significantly increased in restimulated splenocyte populations as well as in purified splenic dendritic cell cultures. These results provide evidence that expression of JNK2 plays a role in T. gondii-induced immunopathology, at the same time in promoting susceptibility to this parasitic pathogen. Copyright © 2010. Published by Elsevier Inc.
PMID: 20117109 [PubMed - as supplied by publisher]
Absence of mitogen-activated protein kinase family member c-Jun N-terminal kinase-2 enhances resistance to Toxoplasma gondii
Sukhumavasi W, Warren AL, Alonso LD, Denkers EY.
Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
The function of mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK)-2 in resistance and pathology during infection has not been greatly studied. Here, we employed Jnk2(-/-) mice to investigate the role of JNK2 in resistance and immunity during oral infection with the protozoan pathogen Toxoplasma gondii. We found increased host resistance in the absence of JNK2 as determined by lower parasite burden and increased host survival. Lack of JNK2 also correlated with decreased neutrophil recruitment to the intestinal mucosa and less pathology in the small intestine. In the absence of JNK2, IL-12 production was slightly but significantly increased in restimulated splenocyte populations as well as in purified splenic dendritic cell cultures. These results provide evidence that expression of JNK2 plays a role in T. gondii-induced immunopathology, at the same time in promoting susceptibility to this parasitic pathogen. Copyright © 2010. Published by Elsevier Inc.
PMID: 20117109 [PubMed - as supplied by publisher]
Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute Toxoplasmosis
Vaccine. 2010 Jan 28. [Epub ahead of print]
Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute Toxoplasmosis: An interplay of IFN-gamma and cytolytic T cells
Jongert E, Lemiere A, Van Ginderachter J, De Craeye S, Huygen K, D'Souza S.
Laboratory for Toxoplasmosis, Pasteur Institute of Brussels, Brussels, Belgium.
Development of prophylactic vaccines against Toxoplasma gondii is based on the observation that latently infected subjects are protected against secondary infection during pregnancy. Cocktail DNA vaccines have been shown to provide high resistance to parasite challenge, and latently infected mice are protected against acute disease. In order to characterize the associated Th1 cellular immune responses in vivo, we used H2-K(k) bone marrow macrophage cell lines constitutively expressing T. gondii GRA1, GRA7 or ROP2 antigens, for the in vivo characterization of antigen-specific T cells in an antigenic challenge model, and as target cells in an in vivo CTL assay. In latently infected C3H/HeN mice, CD4(+) and CD8(+) T cells were recruited to the peritoneal cavity after i.p. challenge with these syngeneic cell lines. GRA1 and GRA7-specific T cells from infected mice were IFN-gamma(+) FasL(-) CD107(-). No IFN-gamma or lytic markers were observed against ROP2. In cocktail DNA vaccinated C3H/HeN mice, the response was restricted to GRA1-specific CD8(+) IFN-gamma(-) FasL(-) CD107(+) T cells. Target cells expressing GRA1 and GRA7, but not ROP2, were efficiently killed in an in vivo CTL assay in latently infected mice, while in DNA vaccinated mice only lysis of GRA1 expressing target cells was observed. Both forms of immunization, DNA vaccination and latent infection, completely protected mice against acute Toxoplasmosis. The results obtained in this work suggest that distinct in vivo cytolytic effector mechanisms are at work in DNA vaccinated and latently infected mice, but both converge to protect against acute toxoplasmosis. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20117266 [PubMed - as supplied by publisher]
Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute Toxoplasmosis: An interplay of IFN-gamma and cytolytic T cells
Jongert E, Lemiere A, Van Ginderachter J, De Craeye S, Huygen K, D'Souza S.
Laboratory for Toxoplasmosis, Pasteur Institute of Brussels, Brussels, Belgium.
Development of prophylactic vaccines against Toxoplasma gondii is based on the observation that latently infected subjects are protected against secondary infection during pregnancy. Cocktail DNA vaccines have been shown to provide high resistance to parasite challenge, and latently infected mice are protected against acute disease. In order to characterize the associated Th1 cellular immune responses in vivo, we used H2-K(k) bone marrow macrophage cell lines constitutively expressing T. gondii GRA1, GRA7 or ROP2 antigens, for the in vivo characterization of antigen-specific T cells in an antigenic challenge model, and as target cells in an in vivo CTL assay. In latently infected C3H/HeN mice, CD4(+) and CD8(+) T cells were recruited to the peritoneal cavity after i.p. challenge with these syngeneic cell lines. GRA1 and GRA7-specific T cells from infected mice were IFN-gamma(+) FasL(-) CD107(-). No IFN-gamma or lytic markers were observed against ROP2. In cocktail DNA vaccinated C3H/HeN mice, the response was restricted to GRA1-specific CD8(+) IFN-gamma(-) FasL(-) CD107(+) T cells. Target cells expressing GRA1 and GRA7, but not ROP2, were efficiently killed in an in vivo CTL assay in latently infected mice, while in DNA vaccinated mice only lysis of GRA1 expressing target cells was observed. Both forms of immunization, DNA vaccination and latent infection, completely protected mice against acute Toxoplasmosis. The results obtained in this work suggest that distinct in vivo cytolytic effector mechanisms are at work in DNA vaccinated and latently infected mice, but both converge to protect against acute toxoplasmosis. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20117266 [PubMed - as supplied by publisher]
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