Syamalima Dube

Degenerate and specific PCR assays for the detection of bovine leukaemia virus and primate T cell leukaemia/lymphoma virus pol DNA and RNA: phylogenetic comparisons of amplified sequences from cattle and primates from around the world

Degenerate and specific PCR assays were developed for bovine leukaemia virus (BLV) and/or primate T cell leukaemia/lymphoma viruses (PTLV). The degenerate assays detected all major variants of the BLV/PTLV genus at a sensitivity of 10-100 copies of input DNA; the specific systems detected 1-10 copies of input target. Sensitivity was 100% in specific DNA-PCR assays done on peripheral blood from seropositive BLV-infected cattle and HTLV-I- or HTLV-II-infected humans, and 62% in RNA/DNA-PCR assays on sera from BLV seropositive cattle. The pol fragments from 21 different BLV strains, isolated from cattle in North and Central America, were cloned and sequenced, and compared to other published BLV and PTLV pol sequences. BLV and PTLV sequences differed by 42%. Sequence divergence was up to 6% among the BLV strains, and up to 36% among the PTLV strains (with PTLV-I and PTLV-II differing among themselves by 15% and 8%, respectively). Some cows were infected with several BLV strains. Among retroviruses, BLV and PTLV sequences formed a distinct clade. The data support the interpretation that BLV and PTLV evolved from a common ancestor many millennia ago, and some considerable time before the PTLV-I and PTLV-II strains diverged from each other. The dissemination of the BLV strains studied probably resulted from the export of European cattle throughout the world over the last 500 years. The relatively similar mutation rates of BLV and PTLV, after their various points of divergence, suggest that there could be a much wider genetic range of BLV than has currently been defined.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection

BACKGROUND: HTLV‐I and HTLV‐II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV‐I antigens have been used to screen for the presence of HTLV‐I or ‐II antibodies. The HTLV‐I‐based assays have not been as sensitive in detecting antibodies to HTLV‐II as in detecting antibodies to HTLV‐I. The Abbott HTLV‐I/HTLV‐II ELISA uses a combination of HTLV‐I and HTLV‐II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV‐I‐based serologic assays and an HTLV‐II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV‐II is endemic.

Characterization of a TM‐4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl

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 ∼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. J. Cell. Biochem. 85: 747–761, 2002.

Tropomyosin expression and dynamics in developing avian embryonic muscles

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.

Identification, characterization, and expression of a novel α‐tropomyosin isoform in cardiac tissues in developing chicken

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 α‐Tm tropomyosin isoform, designated as α‐Tm‐2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (α‐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 α‐Tm‐1 and α‐Tm‐2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of α‐Tm‐2 in embryonic and adult skeletal muscle, however, the α‐Tm‐1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the α‐TM gene, i.e., α‐smooth muscle tropomyosin (α‐Sm), α‐Fibroblast‐1 (α‐F1), and α‐Fibroblast‐2 (α‐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 α‐Tm‐1 and α‐Tm‐2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the α‐TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

The complete genomic sequence of an in vivo low replicating BLV strain

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 41s deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ 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 TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010.

A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture.

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/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.

Anti‐sense‐mediated inhibition of expression of the novel striated tropomyosin isoform TPM1κ disrupts myofibril organization in embryonic axolotl hearts

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 (TPM1κ). TPM1κ 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 TPM1κ and in vitro analysis confirmed the specificity of the TPM1κ anti‐sense oligonucleotide. Altered expression of the novel TM isoform disrupted myofibril structure and function in embryonic hearts.

endemic Infection With Htlv-iib in Venezuelan Indians: Molecular Characterization

The peripheral blood of 41 Yaruro and Guahibo Indians from Venezuela was examined for HTLV antibodies and DNA. Twenty-five samples (61%) were found to be infected with HTLV-IIB. The sensitivities of the serologic and DNA polymerase chain reaction (PCR) analyses were 80% and 96%, respectively. Epidemiologic studies supported both sexual and perinatal transmission of the virus. Sequence analyses of the HTLV-IIB strains from these Indians indicate that they are unique relative to HTLV-II detected in other groups of humans. HTLV-IIB-G2 isolated from a Guahibo Indian is the most divergent HTLV-IIB strain relative to the prototype HTLV-II NRA.