[show abstract][hide abstract] ABSTRACT: Simian T-cell lymphoma/leukemia virus-1 (STLV-1) infection of non-human primates can serve as a model for human T-cell lymphoma/leukemia virus infection.
Two tantalus and 2 patas monkeys were transfused with intraspecies whole blood infected with STLV-1. Infection was determined by ELISA, western blot and DNA PCR analyses. The entire genome of the STLV-1 Tan 90 strain and some of the STVL-1 Pat74 strain were amplified using over-lapping primer-pairs and subsequently sequenced.
Followup studies conducted over 2 years indicated that all 4 monkeys remained healthy despite being infected with STLV-1, as determined by PCR, cloning and sequencing analyses. ELISA and Western blot analyses indicated that both patas monkeys seroconverted within 2 months of transfusion, while one tantalus monkey required one year to seroconvert and the other never fully seroconverted. The tantalus monkey which never fully seroconverted, failed to react to HTLV-1 p24 Gag antigen. Sequence analyses indicated that, while unique, the deduced p24 Gag amino acid sequence of the STLV-1 Tan 90 strain used for infection was still highly homologous to the HTLV-1 p24 Gag amino acids present in the ELISA and WB assays. However, a mutation in the pol sequence of STLV-1 Tan 90 encoded a putative stop codon, while a common deletion in the pol/rex regulatory gene causes significant changes in the Pol, and p27 Rex proteins. These same mutations were also observed in the viral DNA of both recipient infected tantalus monkeys and were not present in the STLV-1 Pat 74 strain.
Our data suggest that seroconversion to STLV-1 infection may be prolonged due to the above mutations, and that compensatory molecular events must have occurred to allow for virus transmission.
[show abstract][hide abstract] ABSTRACT: Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.
[show abstract][hide abstract] ABSTRACT: TPM1kappa is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1kappa expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1kappa protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1kappa mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1kappa specific antibody, we demonstrated the expression and incorporation of TPM1kappa protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1kappa in myofibrillogenesis and sarcomeric function.
Journal of Cellular Biochemistry 07/2010; 110(4):875-81. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Samples were obtained from 53 large granular lymphocytic leukemia (LGLL) patients and 10,000 volunteer blood donors (VBD). Sera were screened in an HTLV-1 enzyme immunoassay (EIA) and further analyzed in peptide-specific Western blots (WB). DNAs were analyzed by HTLV-1, -2, -3, and -4-specific PCR. Forty four percent of LGLL patients vs. 0.12 % of VBD had anti-HTLV antibodies via EIA (p < 0.001). WB and PCR revealed that four LGLL patients (7.5%) vs. one VBD patient (0.01%) were infected with HTLV-2 (p < 0.001), suggesting an HTLV-2 etiology in a minority of cases. No LGLL patient was positive for HTLV-1, -3, or -4, whereas only one EIA-positive VBD was positive for HTLV-1 and none for HTLV-3 or -4. The HTLV EIA-positive, PCR-negative LGLL patients' sera reacted to epitopes within HTLV p24 gag and gp21 env. Other then the PTLV/BLV viruses, human endogenous retroviral element HERV K10 was the only sequence homologous to these two HTLV peptides, raising the possibility of cross-reactivity. Although three LGLL patients (5.7%) vs. none of 110 VBD patients tested positive for antibodies to the homologous HERV K10 peptide (p = 0.03), the significance of the anti-HTLV seroreactivity observed in many LGLL patients remains unclear. Interestingly, out of 36 HTLV-1-positive control subjects, 3 (8%) (p = 0.014) were positive for antibodies to HERV K10; all three had myelopathy. Out of 64 HTLV-2-positive control subjects 16 (25%) (p = <0.001) were positive for HERV K10 antibodies, and 4 (6%) of these had myelopathy. Out of 22 subjects with either HTLV-1 or -2 myelopathy, 7 (31.8%) were positive for HERV K10 antibodies, and out of 72 HTLV-infected subjects without myelopathy, 12 (16.7%) were positive for anti-HERV K10 antibodies (p = 0.11). The prevalence of anti-HERV K10 antibodies in these populations and the clinical implications thereof need to be pursued further.
AIDS research and human retroviruses 01/2010; 26(1):33-40. · 2.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA was extracted from lamb lymphocytes that were infected in vivo with a BLV strain after inoculation with the peripheral blood mononuclear cells from a persistently sero-indeterminate, low viral load, BLV-infected Holstein cow (No. 41) from Argentina. The DNA was PCR amplified with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 41 DNA was cloned, sequenced, and compared phylogenetically to other BLV sequences including an in vivo high replicating strain (BLV ARG 38) from the same herd in Argentina. Characterization of BLV ARG 41's deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.
[show abstract][hide abstract] ABSTRACT: Nkx2.5, a homeodomain-containing transcription factor, is known to be necessary for normal heart development in vertebrates. It is one of the earliest lineage-restricted genes expressed in cardiovascular progenitor cells and knowledge of its expression patterns has important therapeutic implications for damaged cardiomyocytes. Mexican axolotl is a unique system to study heart development for two reasons: the presence of a mutant phenotype lacking organized myofibrils due to sarcomeric tropomyosin deficiency and the ability to induce metamorphosis by administration of exogenous thyroid hormone. In this study, we cloned and sequenced the as yet uncharacterized Nkx2.5 cDNA from normal and cardiac mutant axolotl heart RNA. Comparison of cDNA sequences of Nkx2.5 from normal and mutant axolotl hearts did not show differences suggesting that loss of function mutation in Nkx2.5 is not responsible for the mutant phenotype. However, quantitative studies show higher expression of Nkx2.5 in mutant hearts raising the possibility that increased expression of Nkx2.5 may contribute to the mutant phenotype. We also evaluated quantitative changes in expression of Nkx2.5 in axolotl hearts during embryonic and postembryonic heart development induced by exogenous thyroid hormone. There is an apparent increase in Nkx2.5 transcript levels in metamorphosed hearts.
[show abstract][hide abstract] ABSTRACT: The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.
Cell Motility and the Cytoskeleton 06/2008; 65(5):379-92. · 4.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene "c", for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac troponin T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart.
Biochemical and Biophysical Research Communications 06/2007; 357(1):32-7. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin, a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a later stage. The formation of functional sarcomeres coincided with contractions in normal hearts at stage 35. A-bands and I-bands were formed at stage 35 and did not change at stage 39. The widening of Z-bodies into z-lines was the main developmental difference between stage 35 and 39 normal hearts. Relative to normal hearts, a reduction of sarcomeric protein levels in mutant hearts at stage 35 was found, and a greater reduction occurred at later stages. The lower level of tropomyosin limited the areas where organized myofibrils formed in the mutant. The areas that had tropomyosin staining also had staining for alpha-actinin and myosin. Early myofibrils formed in these areas but the A-bands and I-bands were shorter than normal. At a later stage in the mutant, A-bands and I-bands remained shorter and importantly the Z-bodies also did not form wider z-lines.
[show abstract][hide abstract] ABSTRACT: The Mexican axolotl, Ambystoma mexicanum, serves as an intriguing model to investigate myofibril organization and heart development in vertebrates. The axolotl has a homozygous recessive cardiac lethal gene "c" which causes a failure of ventricular myofibril formation and contraction. However, the conus of the heart beats, and has organized myofibrils. Tropomyosin (TM), an essential component of the thin filament, has three known striated muscle isoforms (TPM1alpha, TPM1kappa, and TPM4alpha) in axolotl hearts. However, it is not known whether there are differential expression patterns of these tropomyosin isoforms in various segments of the heart. Also, it is not understood whether these isoforms contribute to myofibril formation in a segment-specific manner. In this study, we have utilized anti-sense oligonucleotides to separately knockdown post-transcriptional expression of TPM1alpha and TPM4alpha. We then evaluated the organization of myofibrils in the conus and ventricle of normal and cardiac mutant hearts using immunohistochemical techniques. We determined that the TPM1alpha isoform, a product of the TPM1 gene, was essential for myofibrillogenesis in the conus, whereas TPM4alpha, the striated muscle isoform of the TPM4 gene, was essential for myofibrillogenesis in the ventricle. Our results support the segmental theory of vertebrate heart development.
Journal of Cellular Biochemistry 11/2006; 99(3):952-65. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the axolotl, Ambystoma mexicanum, a simple, recessive cardiac-lethal mutation in gene "c" results in the hearts of c/c homozygous animals being deficient in sarcomeric tropomyosin (TM) and failing to form mature myofibrils. Subsequently, the mutant hearts do not beat. A three-step model of myofibril assembly recently developed in cell culture prompted a reassessment of the myofibril assembly process in mutant hearts using a relatively new late marker for thin filament assembly, tropomodulin (Tmod). This is, to the best of our knowledge, the first report of tropomodulin in an amphibian system. Tropomodulin antibodies were immunolocalized to the ends of the thin filaments. Tropomodulin was also found in discrete punctate spots in normal and mutant hearts, often in linear arrays suggestive of early myofibril formation. The tropomodulin spots assessed in stage 41/42 mutant hearts co-localized with antibodies to other myofibrillar proteins indicative of nascent myofibril formation. This suggests a failure of elongation/maturation of nascent myofibrils, which could be a consequence of decreased TM levels or increased Tmod/ TM ratio. Unlike tropomyosin, there is no apparent decrease in the level of Tmod expression in mutant hearts.
[show abstract][hide abstract] ABSTRACT: Striated muscle tropomyosin (TM) is described as containing ten exons; 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. Exon 9a/b has critical troponin binding domains and is found in striated muscle isoforms. We have recently discovered a smooth (exon 2a)/striated (exons 9a/b) isoform expressed in amphibian, avian, and mammalian hearts, designated as an isoform of the TPM1 gene (TPM1kappa). TPM1kappa expression was blocked in whole embryonic axolotl heart by transfection of exon-specific anti-sense oligonucleotide. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed lower transcript expression of TPM1kappa and in vitro analysis confirmed the specificity of the TPM1kappa anti-sense oligonucleotide. Altered expression of the novel TM isoform disrupted myofibril structure and function in embryonic hearts.
Journal of Cellular Biochemistry 08/2005; 95(4):840-8. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although the role of tropomyosin is well-defined in striated muscle, the precise mechanism of how tropomyosin functions is still unclear. It has been shown that extension of either N- or C-terminal ends of sarcomeric tropomyosin do not affect cardiac myofibrillogenesis, but it is not known whether simultaneous extension of both ends affects the process. For studying structural/functional relationships of sarcomeric tropomyosin, we have chosen the Ambystoma mexicanum because cardiac mutant hearts are deficient in sarcomeric tropomyosin. In this study, we have made an expression construct, pEGFP.TPM4alpha.E-L-FLAG, that, on transfection into normal and mutant axolotl hearts in organ culture, expresses GFP.TPM4alpha.E-L-FLAG fusion protein in which both the N- and C-termini of TPM4alpha are being extended. TPM4alpha is one of the three tropomyosins expressed in normal axolotl hearts. Both confocal and electron microscopic analyses show that this modified sarcomeric tropomyosin can form organized myofibrils in axolotl hearts.
[show abstract][hide abstract] ABSTRACT: We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.
[show abstract][hide abstract] ABSTRACT: Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non-muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM-4 type. This novel alpha-Tm tropomyosin isoform, designated as alpha-Tm-2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (alpha-Tm-1) was also expressed in embryonic hearts along with the striated muscle isoform of TM-4. In adult heart, TM-4 was expressed, however, expression of both alpha-Tm-1 and alpha-Tm-2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of alpha-Tm-2 in embryonic and adult skeletal muscle, however, the alpha-Tm-1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the alpha-TM gene, i.e., alpha-smooth muscle tropomyosin (alpha-Sm), alpha-Fibroblast-1 (alpha-F1), and alpha-Fibroblast-2 (alpha-F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of alpha-Tm-1 and alpha-Tm-2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the alpha-TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.
Journal of Cellular Biochemistry 07/2003; 89(3):427-39. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serologic evidence of Trypanosoma cruzi infection was demonstrated in 43.5% of 519 Paleoamerindians and in only 2.5% of 161 non-Indians (Mennonites of German descent and Paraguayans of Spanish descent) inhabiting an area of western Paraguay that belongs to the Gran Chaco territory. These people ranged in age between two and 80 years. All were also tested for infection with the human T cell lymphotropic virus type II (HTLV-II). The prevalence of HTLV-II infection was 22.1% in Indians and 3.7% in non-Indians. As determined by a multivariate logistic regression analysis that controlled for relevant confounders, an HTLV-II-infected individual was 2.28 times more likely to be seropositive for T. cruzi than an HTLV-II negative. Possible explanations for this finding are discussed. The difference in T. cruzi prevalence between Indians and non-Indians was associated with differences between these groups in exposure to known risk factors for infection with the parasite. There were significant differences in the seroprevalence of T. cruzi among the two predominant Indian groups, even when they inhabited communities that were close to each other. These differences were associated with differences in the prevalence of HTLV-II infection but not with differences in exposure to known risk factors for T. cruzi infection. Infection with T. cruzi increased with age, was greater in males than in females, and clustered in families.
The American journal of tropical medicine and hygiene 03/2003; 68(2):235-41. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/striated chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.
[show abstract][hide abstract] ABSTRACT: A striated muscle isoform of a Tropomyosin (TM-4) gene was characterized and found to be necessary for contractile function in embryonic heart. The full-length clone of this isoform was isolated from the Mexican axolotl (Ambystoma mexicanum) and named Axolotl Tropomyosin Cardiac-3 (ATmC-3). The gene encoded a cardiac-specific tropomyosin protein with 284 amino acid residues that demonstrated high homology to the Xenopus cardiac TM-4 type tropomyosin. Northern blot analysis indicates a transcript of approximately 1.25 kb in size. RT-PCR and in situ hybridization demonstrated that this isoform is predominantly in cardiac tissue. Our laboratory uses an animal model that carries a cardiac lethal mutation (gene c), this mutation results in a greatly diminished level of tropomyosin protein in the ventricle. Transfection of ATmC-3 DNA into mutant hearts increased tropomyosin levels and promoted myofibrillogenesis. ATmC-3 expression was blocked in normal hearts by transfection of exon-specific anti-sense oligonucleotide (AS-ODN). RT-PCR confirmed lower transcript expression of ATmC-3 and in vitro analysis confirmed the specificity of the ATmC-3 exon 2 anti-sense oligonucleotide. These AS-ODN treated hearts also had a disruption of myofibril organization and disruption of synchronous contractions. These results demonstrated that a striated muscle isoform of the TM-4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle.
Journal of Cellular Biochemistry 02/2002; 85(4):747-61. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Classic Kaposi's sarcoma (KS) is rare in children. Although its etiology is not fully understood, human herpesvirus 8 (HHV-8) is present in the angiogenic lesions. We report an HIV-negative, 13-year-old patient of Sicilian descent with HHV-8-associated classic KS to facilitate the diagnosis and treatment of this entity in children.
DNA was extracted from the skin specimen of the patient and analyzed via PCR assay and Southern blot hybridization for HHV-8 DNA. The amplified HHV-8 DNA was cloned, sequenced, and compared with the prototype HHV-8-KS330/BAM.
The patient presented with purpuric lesions on the distal lower extremities and the tip of his nose, associated with thrombocytopenia and leukopenia, suggesting an immune-mediated cytopenia. While on prednisone, he developed marked vascular proliferation in the groins. Biopsy of the skin lesions showed KS, and HHV-8 was detected in the tissues by PCR. Sequence analysis of the amplified DNA was homologous to the prototype HHV-8-KS330/BAM. His HHV-8 strain was the A subgroup, the type associated with Mediterranean classic KS. Stopping prednisone and treatment with IFN-alpha and IgG resulted in regression of the groin lesions.
This report emphasizes the importance of recognizing classic KS in children and avoiding immunosuppressive therapies in indolent classic KS. The diagnostic and therapeutic strategies were effective and well tolerated.
Clinical Cancer Research 09/2001; 7(8):2263-8. · 7.84 Impact Factor