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1.
Mol Cell Biol ; 21(22): 7872-82, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11604521

ABSTRACT

While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions.


Subject(s)
DNA-Binding Proteins/genetics , Protozoan Proteins/genetics , Transcription Initiation Site , Trichomonas vaginalis/genetics , Trichomonas vaginalis/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Chromatography, Affinity/methods , Cloning, Molecular , DNA, Protozoan , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Eukaryotic Cells , Molecular Sequence Data , Protozoan Proteins/isolation & purification , Protozoan Proteins/metabolism
2.
J Parasitol ; 87(1): 19-23, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11227890

ABSTRACT

Relatively little information is available concerning the expression of parasite genes during the liver stage of Plasmodium infection, mostly because of low-level infection of host hepatocytes and the lack of purification techniques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To determine which route was optimal, BALB/c mice were inoculated via 1 of these routes, and parasitemia was detected by reverse transcription polymerase chain reaction (RT-PCR) detecting both murine beta-actin and P. yoelii-specific 28S ribosomal RNA in the liver samples. Murine beta-actin was detected after 15 cycles of PCR, and its expression levels did not differ between treatment groups. However, P. yoelii-specific 28S ribosomal RNA gene product was detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahepatic portal circulation. Experiments were then performed to identify the smallest inoculum required to initiate a liver stage infection that would yield sufficient parasite RNA for analysis. Inoculation with different doses of sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first detected in both treatment groups after 15 cycles, suggesting that both doses of sporozoite inocula provided relatively the same level of liver infection rate. We also have analyzed infected mouse liver for parasite-specific mRNA, which was detectable as early as 24 hr postinfection.


Subject(s)
Antigens, Protozoan , Gene Expression , Hepatocytes/parasitology , Malaria/parasitology , Plasmodium yoelii/growth & development , Plasmodium yoelii/genetics , RNA, Messenger/metabolism , Animals , Female , Gene Expression Profiling , Mice , Mice, Inbred BALB C , Plasmodium yoelii/isolation & purification , Plasmodium yoelii/pathogenicity , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA, Messenger/genetics , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , RNA, Ribosomal, 28S/genetics , RNA, Ribosomal, 28S/metabolism , Reverse Transcriptase Polymerase Chain Reaction
3.
J Immunol ; 166(3): 1945-50, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11160243

ABSTRACT

The liver stage of malaria, caused by the genus Plasmodium, is clinically silent, but immunologically significant. Ample evidence exists for an effective CD8(+) T cell response to this stage as well as the involvement of gammadeltaT cells and NK1.1(int) cells in immunized animal models. In contrast, there is little information concerning responses in a naive host. Here we report that several host gene expressions in the liver, spleen, and kidney of BALB/c mice are altered during the liver stage of Plasmodium yoelii infection. Really interesting new gene 3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase, and p45 NF erythroid 2 were all up-regulated 24 h after infection with P. yoelii. Semaphorin subclass 4 member G expression was elevated in the kidney, whereas Ring3 was elevated in both spleen and kidney. The expression of TNF-alpha (TNF-alpha and IFN-gamma) were down-regulated in all three tissues tested except in infected spleen where IFN-gamma was elevated. P. yoelii-related host gene changes were compared with those in Toxoplasma gondii-infected livers. Ring3 expression increased 5-fold over control values, whereas expression of the other transcripts remained unchanged. TNF-alpha and IFN-gamma expressions were increased in the Toxoplasma-infected livers. The uniform increase of Ring3 expression in both Plasmodium- and Toxoplasma-infected livers suggests an innate immune response against parasitic infections, whereas the other gene expression changes are consistent with Plasmodium parasite-specific responses. Taken together, these changes suggest the immune responses to P. yoelii infection are both parasite and organ specific.


Subject(s)
Liver/immunology , Liver/parasitology , Malaria/immunology , Malaria/parasitology , Plasmodium yoelii/growth & development , Plasmodium yoelii/immunology , Semaphorins , Animals , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , Female , Gene Expression Regulation, Developmental/immunology , Host-Parasite Interactions , Malaria/enzymology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , NF-E2 Transcription Factor, p45 Subunit , Nerve Growth Factors/biosynthesis , Nerve Growth Factors/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Organ Specificity/genetics , Organ Specificity/immunology , Plasmodium yoelii/genetics , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
4.
Mol Biochem Parasitol ; 111(1): 143-51, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11087924

ABSTRACT

Differential display (DD) has been routinely used to identify genes whose expression pattern is altered by changes in the cellular environment and/or at different stages of development. Most reports utilizing DD contain conventional DD primers that have high guanine and cytosine content and would not be expected to be optimal for Plasmodium which has approximately 30-40% G+C. In an attempt to accommodate the high adenine and thymidine rich genome of Plasmodium yoelii, we utilized PCR primers containing 40, 50 and 60% G+C and modified the existing DD technique. Thus 40% G+C appeared to be the most suitable to amplify Plasmodium genome. Gene specific primers were generated from the sequences of selected DD bands amplified using the 40% G+C primers and were used to verify that the DD clones were of parasite origin by PCR and sequence alignment. Additional data on five of the selected DD clones, designated P2T1L5, P2T1L6, P2T6L11, P2T7L12 and P2T7L13, suggested that all are expressed during the P. yoelii liver stage infection. Interestingly, P2T1L5 is also expressed during the sporozoite stage of the life cycle and both P2T1L6 and P2T6L11 are present as blood stage antigens. The results of this study suggest that DD incorporating primers with low G+C content allows the identification of P. yoelii messages from infected mouse livers.


Subject(s)
Gene Expression Profiling , Gene Expression , Liver/parasitology , Malaria/parasitology , Plasmodium yoelii/genetics , Animals , Base Composition , DNA Primers , DNA, Complementary , Female , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Plasmodium falciparum/genetics , Plasmodium yoelii/growth & development , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism
5.
Infect Immun ; 67(11): 6104-8, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10531273

ABSTRACT

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.


Subject(s)
Bacterial Proteins , Genes, Bacterial , Hemolysin Proteins/genetics , Rickettsia typhi/genetics , Amino Acid Sequence , Animals , Chlorocebus aethiops , Cloning, Molecular , Hemolysin Proteins/toxicity , Molecular Sequence Data , Polymerase Chain Reaction , Sheep , Transformation, Bacterial , Vero Cells
6.
Blood ; 86(8): 3196-204, 1995 Oct 15.
Article in English | MEDLINE | ID: mdl-7579415

ABSTRACT

During intraerythrocytic growth of Plasmodium falciparum, several parasite proteins are transported from the parasite to the erythrocyte membrane, where they bind to membrane skeletal proteins. Mature-parasite-infected erythrocyte surface antigen (MESA) has previously been shown to associate with host erythrocyte membrane skeletal protein 4.1. Using a spontaneous mutant of P falciparum that has lost the ability to synthesize MESA and 4.1-deficient erythrocytes, we examined growth of MESA(+) and MESA(-) parasites in normal and 4.1-deficient erythrocytes. Viability of MESA(+) parasites was reduced in 4.1-deficient erythrocytes as compared with that for normal erythrocytes, but MESA(-) parasites grew equally well in 4.1-deficient and normal erythrocytes. Cytoadherence of MESA(+)- and MESA (-)-parasitized normal and 4.1-deficient erythrocytes to C32 melanoma cells was similar, indicating that neither protein 4.1 nor MESA plays a major role in cytoadherence of infected erythrocytes. Localization of MESA in normal and 4.1-deficient erythrocytes was examined by confocal microscopy. MESA was diffusely distributed in the cytosol of 4.1-deficient erythrocytes but was membrane-associated in normal erythrocytes. These findings suggest that MESA binding to protein 4.1 plays a major role in intraerythrocytic parasite viability.


Subject(s)
Antigens, Protozoan/physiology , Cytoskeletal Proteins , Erythrocytes/parasitology , Malaria, Falciparum/blood , Membrane Proteins/metabolism , Neuropeptides , Plasmodium falciparum/physiology , Protozoan Proteins/physiology , Animals , Cell Adhesion , Host-Parasite Interactions , Melanoma/pathology , Membrane Proteins/deficiency , Microscopy, Confocal , Peptides/physiology , Plasmodium falciparum/immunology , Protein Binding , Rabbits , Tumor Cells, Cultured
7.
Proc Soc Exp Biol Med ; 207(2): 220-6, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7938053

ABSTRACT

The studies reported herein were conducted to confirm that the pituitary gland is involved in maintaining growth hormone (GH) resistance in rats with insulin-dependent diabetes mellitus (IDDM) and to determine whether the adrenocorticotropic hormone (ACTH)-adrenal cortical axis is responsible. The rats were made diabetic by injecting streptozotocin (85 mg/kg body wt) IP once daily on two consecutive days. They were then injected with 15 IU insulin SC twice daily on two consecutive days to enable them to survive hypophysectomy or adrenalectomy. Intact nondiabetic (NonDb), diabetic (Db), hypophysectomized diabetic (HxDb), and adrenalectomized diabetic (AxDb) rats were injected twice daily with 50 micrograms porcine (p) GH or with 0.9% saline for 2 weeks following the surgeries. Serum glucose levels of the saline-injected Db, HxDb, and AxDb rats were significantly greater than those of the NonDb rats by 106%, 65% and 49%, respectively. However, the levels in the HxDb and AxDb animals were significantly lower than those of the Db group by 20% and 28%, respectively. Injections of pGH into NonDb rats increased serum glucose concentrations by 38%, over their saline-treated controls, and by 29% in AxDb rats. This diabetogenic effect of GH was not seen in any other group. Administration of pGH to Db rats failed to increase body weight gain, tall growth, tibial epiphysial plate width, or serum IGF-I concentration over saline-injected controls. By contrast, HxDb and AxDb rats injected with pGH showed significant increases in all four growth parameters. Total serum IGF-I concentrations in AxDb rats injected with pGH equaled those in NonDb controls. To determine whether the lack of corticosterone (B) in the AxDb rats was responsible for the reduced hyperglycemia and restored responsiveness to pGH, AxDb rats were given B in their drinking water at 5 or 25 micrograms/ml. Administration of B reduced the beneficial effects of adrenalectomy by restoring hyperglycemia and growth impairment, and partially restored resistance to the pGH injections. These studies confirm that the pituitary contributes to diabetic growth impairment and show that the ACTH-adrenal cortical axis is primarily responsible for the GH-resistant state that develops in rats with IDDM.


Subject(s)
Adrenalectomy , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Growth Hormone/pharmacology , Hypophysectomy , Animals , Blood Glucose/analysis , Corticosterone/pharmacology , Insulin-Like Growth Factor I/analysis , Male , Rats , Streptozocin , Weight Gain/drug effects
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