[Show abstract][Hide abstract] ABSTRACT: Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, μ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.
[Show abstract][Hide abstract] ABSTRACT: We evaluated the effect of shz-1, a cardiogenic molecule, on the expression of various tropomyosin (TM) isoforms in the Mexican axolotl (Ambystoma mexicanum) hearts. qRT-PCR data show a ~1.5-fold increase in cardiac transcripts of the Nkx2.5 gene, which plays a crucial role in cardiogenesis in vertebrates. Shz-1 augments the expression of transcripts of the total sarcomeric TPM1 (both TPM1α & TPM1κ) and sarcomeric TPM4α. In order to understand the mechanism by which shz-1 augments the expression of sarcomeric TPM transcription in axolotl hearts, we transfected C2C12 cells with pGL3.axolotl. We transfected C2C12 cells with pGL3-axolotl TPM4 promoter constructs containing the firefly luciferase reporter gene. The transfected C2C12 cells were grown in the absence or presence of shz-1 (5 μM). Subsequently, we determined the firefly luciferase activity in the extracts of transfected cells. The results suggest that shz-1 activates the axolotl TPM4 promoter-driven ectopic expression in C2C12 cells. Also, we transfected C2C12 cells with a pGL3.1 vector containing the promoter of the mouse skeletal muscle troponin-I and observed a similar increase in the luciferase activity in shz-1-treated cells. We conclude that shz-1 activates the promoters of a variety of genes including axolotl TPM4. We have quantified the expression of the total sarcomeric TPM1 and observed a 1.5-fold increase in treated cells. Western blot analyses with CH1 monoclonal antibody specific for sarcomeric isoforms show that shz-1 does not increase the expression of TM protein in axolotl hearts, whereas it does in C2C12 cells. These findings support our hypothesis that cardiac TM expression in axolotl undergoes translational control.
No preview · Article · Jun 2014 · Cardiovascular Toxicology
[Show abstract][Hide abstract] ABSTRACT: We have investigated the expression of TPM1 α and TPM1 κ in mouse striated muscles. TPM1 α and TMP1 κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1 κ . However, the deduced amino acid sequences of human TPM1 α and mouse TPM1 α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1 κ is significantly lower compared to TPM1 α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1 κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1 κ in mammalian skeletal muscle.
[Show abstract][Hide abstract] ABSTRACT: The anatomy, function and embryonic development of the heart have been of interest to clinicians and researchers alike for centuries. A beating heart is one of the key criteria in defining life or death in humans. An understanding of the multitude of genetic and functional elements that interplay to form such a complex organ is slowly evolving with new genetic, molecular and experimental techniques. Despite the need for ever more complex molecular techniques some of our biggest leaps in knowledge come from nature itself through observations of mutations that create natural defects in function. Such a natural mutation is found in the Mexican axolotl, Ambystoma mexicanum. It is a facultative neotenous salamander well studied for its ability to regenerate severed limbs and tail. Interestingly it also well suited to studying segmental heart development and differential sarcomere protein expression due to a naturally occurring mendelian recessive mutation in cardiac mutant gene “c”. The resultant mutants are identified by their failure to beat and can be studied for extended periods before they finally die due to lack of circulation. Studies have shown a differential expression of tropomyosin between the conus and the ventricle indicating two different cardiac segments. Tropomyosin protein, but not its transcript have been found to be deficient in mutant ventricles and sarcomere formation can be rescued by the addition of TM protein or cDNA. Although once thought to be due to endoderm induction our findings indicate a translational regulatory mechanism that may ultimately control the level of tropomyosin protein in axolotl hearts.
[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.
No preview · Article · May 2011 · Cardiovascular toxicology
[Show abstract][Hide abstract] ABSTRACT: Seventeen chemotherapy-induced cardiomyopathy (CM) patients and 100 volunteer blood donors (VBD) were analyzed for genetic variability in the following sarcomeric protein genes: cardiac alpha actin (ACTC); myosin light chain 2 (MYL2); myosin light chain 3 (MYL3); myosin heavy chain 7 (MYH7); myosin binding protein C3 (MYBPC3); tropomyosin 1 (TPM1); cardiac troponin I (TNNI3); and cardiac troponin T (TNNT2). No previously published mutations associated with familial CM were found in any of the subjects. No differences in the subjects and the published literature were found in the ACTC, MYL2, or TNNI3 genes. However, eight single nucleotide polymorphisms were identified in the other genes analyzed: a base change in intron 1a of TPM1; a synonymous change in exon 4 of TPM1; a base change in intron 11 of TNNT2; base changes in introns 2 and 4 of MYL3; a base change in introns 8 and 11 of MYH7; and a synonymous change in exon 31 of MYPC3. Both homozygous and heterozygous genotypes for each of these areas were identified among the subjects. Certain genotypes occurred more frequently in the CM patients and were deemed to be "skewed", with the MYH7I8 (TT) genotype approaching statistical significance (p = 0.052). Eighty-eight percent of the CM patients had one or more "skewed" genotypes, while only 64 percent of the VBD had this genetic pattern (p = 0.032). An increasing number of "skewed" genotypes was significantly associated with cardiomyopathy. Certain genotypes did not occur at all among the CM patients, and theoretically may confer protection against chemotherapy induced toxicity. Only one subject, a woman who developed CM on trastuzumab, had the genotype MYL3I2 (T/T), MYL3I4 (C/C), MYH7I11 (CC). Additional in vitro andclinical studies are warranted to understand the phenotypic, physiologic, and clinical significances of these variabilities in the sarcomeric protein genes.
No preview · Article · Jan 2011 · Internet Journal of Cardiology
[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.
[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.
No preview · Article · Jan 2010 · AIDS research and human retroviruses
[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.
No preview · Article · Mar 2009 · Cardiovascular toxicology
[Show abstract][Hide abstract] ABSTRACT: Ninety three years after Siedamgrotzky first reported cases of bovine lymphocytic malignancies in Germany in 1876, bovine leukemia virus (BLV) was identified in cultured neoplastic cells from affected cattle. Epidemiological studies carried out in herds with a high frequency of mature B-cell lymphoma/leukemia proved that BLV was the causative agent of these diseases and also of persistent lymphocytosis (PL). This latter condition results in the benign expansion of BLV-infected peripheral blood B-cell lymphocytes. BLV is identified in almost every case of bovine B-cell lymphoma/leukemia; however, less than 10% of BLV-infected cattle develop these diseases, while about 30% of BLV-infected animals develop PL. Early observations of the increased incidence of Bcell lymphocytic neoplasias or PL in certain bovine pedigrees, but not in others, suggested that host-genetic factors were involved in the development of these conditions after BLV-infection. There were also indications that host-genetic factors influenced the susceptibility to one condition or the other independently. Epidemiological data also indicate that 2/3 of all cases of B-cell neoplasia occur in cattle with pre-existing PL, while 1/3 of cases come from animals with a normal blood profile. The major histocompatibility complex of cattle, termed Bovine Lymphocyte Antigens (BoLA) was discovered in 1978. The BoLA complex is a multiallelic or polymorphic genetic region located on chromosome 23 at band 22, and is organized into class I, II, and III genes, which encode proteins with different tissue distributions and functions. B-lymphocytic leukemia/lymphoma, PL and even the number of BLV-infected peripheral blood lymphocytes were subsequently found to be associated with certain BoLA-genotypes. Due to the wide dissemination of BLV among dairy cattle in Argentina and other countries, control and eradication programs based on the serological detection of BLVinfected cattle and the subsequent culling of infected animals has several drawbacks. A new approach to classify BLV-infected Holstein cattle belonging to high prevalence BLV-infected herds was recently reported. Two BLV-infection profiles: low and high proviral load (LPL and HPL, respectively) showed a strong association with certain BoLA-DRB3.2 polymorphisms. LPL was more frequently associated with BoLADRB3.2* 0902, and HPL with BoLA-DRB3.2*1501 or 03. Further experimentation has indicated that these alleles are good candidates for genetic markers of resistance or susceptibility to BLV transmission, and would serve as powerful tools toward controlling BLV dissemination by marker assisted breeding of genetically-selected cattle. The dynamic between BLV and the BoLA complex should serve as a model for studying the interaction between host and virus. Such understanding should prove important to, not only veterinary medicine, but also to human oncology, wherein human T-cell lymphoma/leukemia virus, a member of the same genus of retroviruses as BLV, causes T-lymphocytic malignancies.
[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.
No preview · Article · May 2008 · Cell Motility and the Cytoskeleton
[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.
Full-text · Article · Jun 2007 · Biochemical and Biophysical Research Communications
[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.
No preview · Article · Feb 2007 · Cardiovascular Toxicology
[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.
No preview · Article · Oct 2006 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Proteins synthesized in vitro by mitochondria isolated from 48-h germinating seeds of Vigna sinensis (L.) Savi and incubated in the presence of 14C-labelled amino acids from Chlorella protein hydrolysate, have been found associated with nine products separable by acrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Cytoplasmic contribution to these products was practically eliminated by the use of cycloheximide. Most of the radioactivity was incorporated into proteins having molecular weights between 10,000 and 65,000 as determined by comparing their electrophoretic mobilities with those of standard, reference proteins.
No preview · Article · Apr 2006 · Physiologia Plantarum