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T Tao

Publications and source records attributed to T Tao.

At least 55 records · Page 3Linked to original sources
PubMed1998

Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.

Adenosine Triphosphatases↗
PubMed1998

Granulocyte colony-stimulating factor (G-CSF) dependent hematopoiesis with monosomy 7 in a patient with severe aplastic anemia after ATG/CsA/G-CSF combined therapy.

We report a case of secondary myelodysplastic syndrome (MDS) with monosomy 7, which evolved from severe aplastic anemia (SAA) after long-term use of granulocyte colony-stimulating factor (G-CSF). A 36 year old female was admitted for detailed examination and treatment of pancytopenia. SAA was diagnosed based on hypoplastic bone marrow and a normal chromosome study. She was treated with anti-thymocyte globulin (ATG), ciclosporin A (CsA) and G-CSF, which resulted in gradual improvement of not only the myeloid but also the erythroid-megakaryocyte series. However, bone marrow dysplasia with monosomy 7 was observed after 7 months of a combination therapy of immunosuppressant and G-CSF, which prompted the discontinuation of G-CSF administration. Thereafter, bone marrow hypoplasia gradually progressed, resulting in a second aplastic crisis. During this process, the proportion of marrow cells showing monosomy 7 decreased, and the proportion with normal karyotype increased. Re-administration of G-CSF induced a trilineage, though dysplastic, hematological response; but the monosomy 7 positive population increased again. These observations indicated the presence of G-CSF dependent hematopoiesis associated with monosomy 7 in this patient. Although many G-CSF related MDS/AML cases with this leukemia-specific abnormal karyotype have been reported with emphasis on the harmful effects of G-CSF, G-CSF was useful even after the appearance of monosomy 7 as a means of avoiding life-threatening infection in this patient.

Adult↗
PubMed1998

Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21.

Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI-TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies.

Amino Acid Sequence↗
PubMed1998

Three amino acid substitutions in the L protein of the human parainfluenza virus type 3 cp45 live attenuated vaccine candidate contribute to its temperature-sensitive and attenuation phenotypes.

Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580-584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43-52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 105-fold reduction in plaque formation in cell culture at 40 degrees C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype of cp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.

Animals↗
PubMed1998

Recovery of a fully viable chimeric human parainfluenza virus (PIV) type 3 in which the hemagglutinin-neuraminidase and fusion glycoproteins have been replaced by those of PIV type 1.

The recent recovery of human parainfluenza virus type 3 (PIV3) from cDNA, together with the availability of a promising, highly characterized live attenuated PIV3 vaccine virus, suggested a novel strategy for the rapid development of comparable recombinant vaccine viruses for human PIV1 and PIV2. The strategy, illustrated here for PIV1, is to create chimeric viruses in which the two protective antigens, the hemagglutinin-neuraminidase (HN) and fusion (F) envelope glycoproteins, of an attenuated PIV3 variant are replaced by those of PIV1 or PIV2. As a first step, this has been achieved by using recombinant wild-type (wt) PIV3 as the recipient for PIV1 HN and F, engineered so that each PIV1 open reading frame is flanked by the existing PIV3 nontranslated regions and transcription signals. This yielded a viable chimeric recombinant virus, designated rPIV3-1, that encodes the PIV1 HN and F glycoproteins in the background of the wt PIV3 internal proteins. There were three noteworthy findings. First, in contrast to recently reported glycoprotein replacement chimeras of vesicular somatitis virus or measles virus, the PIV3-1 chimera replicates in LLC-MK2 cells and in the respiratory tract of hamsters as efficiently as its PIV1 and PIV3 parents. This is remarkable because the HN and F glycoproteins share only 43 and 47%, respectively, overall amino acid sequence identity between serotypes. In particular, the cytoplasmic tails share only 9 to 11% identity, suggesting that their presumed role in virion morphogenesis does not involve sequence-specific contacts. Second, rPIV3-1 was found to possess biological properties derived from each of its parent viruses. Specifically, it requires trypsin for efficient plaque formation in tissue culture, like its PIV1 parent but unlike PIV3. On the other hand, it causes an extensive cytopathic effect (CPE) in LLC-MK2 cultures which resembles that of its PIV3 parent but differs from that of its noncytopathic PIV1 parent. This latter finding indicates that the genetic basis for the CPE of PIV3 in tissue culture lies outside regions encoding the HN or F glycoprotein. Third, it should now be possible to rapidly develop a live attenuated PIV1 vaccine by the staged introduction of known, characterized attenuating mutations present in a live attenuated PIV3 vaccine candidate into the PIV3-1 cDNA followed by recovery of attenuated derivatives of rPIV3-1.

Animals↗
PubMed1998

Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus.

The authors evaluated the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in comparison with virus isolation and detection by an indirect immunoperoxidase assay (VI-IPA). To determine the specificity of the assay, samples from 60 spleens, 45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and four kidneys, collected from pigs of various ages which had been slaughtered in abattoirs in Canada (a population free from CSFV), were tested. All the samples tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples were passaged in porcine kidney (PK) 15 cells and retested by both assays. All were found to be negative, giving a specificity of 100%. To determine the analytical sensitivity of the assay, a similar comparative study was conducted, using CSFV grown in tissue culture and tonsil tissues from a CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per 100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue culture fluids, compared to one infectious unit per ml by VI-IPA. To determine diagnostic sensitivity, three coded panels (two internal and one external), comprising 45 samples from 14 pigs, were tested. The diagnostic sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal panels. The results of the external panel, apart from two samples that were missed by both RT-PCR and VI-IPA, were found to be in total agreement. These two samples remained negative after amplification in PK15 cells. All the RT-PCR results were based on a single test whereas, for the VI-IPA results, positive results were obtained for five samples only after an amplification round in PK15 cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable improved detection of the virus in a shorter time period.

Animals↗
PubMed1997

Expression in insect cells and characterization of the 110 kDa anchoring subunit of myosin light chain phosphatase.

The major myosin light chain phosphatase is composed of three subunits with apparent molecular masses of 130, 38 and 20 kDa, corresponding to the myosin-binding, catalytic and a regulatory subunit of unknown function, respectively. In this work, we have amplified the cDNA coding for each of the three subunits by the polymerase chain reaction, and expressed the 130 kDa subunit in insect cells using the baculovirus expression system. Limited chymotrypsin digestion show that the folding of the expressed protein is similar to that in the native holoenzyme. N-Terminal sequencing reveals that our recombinant protein is authentic. Mass spectrometry shows that the expressed protein is full length. The recombinant protein is capable of binding myosin based on the ELISA assay and myosin affinity chromatography. Finally, rotary shadowing electron microscopy reveals an elongated structure with three globular domains connected by flexible strands. These results pave the way for future biochemical, structural and site-directed mutagenesis studies on the myosin light chain phosphatase. We also found that the cDNA of the 20 kDa subunit may code for a smaller protein with a molecular mass of 18.5 kDa.

Animals↗
PubMed1997

Opposite effects of electrostatics and steric exclusion on bundle formation by F-actin and other filamentous polyelectrolytes.

A number of positively charged polypeptides and proteins bundle DNA, F-actin, microtubules, and viruses such as filamentous phage fd and tobacco mosaic virus (TMV), as well as intermediate filaments formed by vimentin. The general behavior is dictated by the common polyelectrolyte nature of these biopolymers, which gives rise to nonspecific binding by ligands carrying several net opposite charges. An attractive interaction accounts for the subsequent lateral aggregation, distinguishing this transition from the liquid crystalline formation of filamentous particles at high concentrations. Morphologically similar filament bundles can also be induced by inert solutes such as polyethylene glycol (PEG) and proteins that do not bind the macromolecular filaments, but the physicochemistry underlying this class of bundle transitions is distinct. In particular, bundling transitions induced by electrostatic and steric mechanisms have an opposite dependence on the solution ionic strength and the concentration of the filamentous biopolymers. The distinct mechanisms illustrated in this report may each contribute to the formation of specific polymer bundles under physiological conditions.

Actins↗
PubMed1997

Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments.

Contraction of vertebrate striated muscle is regulated by the interaction of Ca2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibitory subunit, responds to the binding of Ca2+ to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N- and C-terminal domains, respectively, of TnI. A mutant rabbit skeletal TnI, TnI48/133 (C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of approximately 41 A for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 A in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca2+ to TnC. In the reconstituted thin filament, this distance remained to be 50 A in the presence of saturating Ca2+, but increased to approximately 66 A on removing Ca2+ with EGTA in the presence of Mg2+. Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca2+-dependent change in the global conformation of TnI in the Tn complex free in solution, the removal of Ca2+ caused a substantial separation of the N- and C-terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the relaxed state.

Actin Cytoskeleton↗
PubMed1997

Association of calponin with desmin intermediate filaments.

Our previous immunoelectron microscopy studies of chicken gizzard smooth muscle cells showed that in certain areas the distribution of anti-calponin exhibits a high degree of overlap with beta-actin, filamin, and in particular, desmin, suggesting that in situ a fraction of calponin may be associated with intermediate filaments of the cytoskeleton. In this work we further explore this idea by studying the interaction between calponin and desmin. We found that at physiological salt concentrations, calponin bound only weakly to synthetic desmin intermediate filaments. On the other hand, calponin bound strongly to nonfilamentous desmin tetramers and was incorporated into intermediate filaments when the two proteins were mixed in a buffer containing 6 M urea and dialyzed into a buffer containing 0.15 M NaCl. Anti-calponin was found to label a portion of intermediate filaments and dense bodies isolated from gizzard tissues. Our findings suggest that in chicken gizzard smooth muscle cells, calponin may be an integral component of desmin intermediate filaments in the vicinity of dense bodies. Since calponin is also known to bind actin, we hypothesize that one of the functions of calponin might be to bridge intermediate filaments with actin in dense bodies.

Animals↗
PubMed1997

Electrostatic effects of smooth muscle calponin on actin assembly.

The contribution of electrostatic interactions to the effects of chicken gizzard calponin on the kinetics of actin polymerization and the bundling of F-actin were characterized by a combination of fluorescence, light-scattering, co-sedimentation, and electron-microscopic methods. Stoichiometric amounts of calponin accelerate actin polymerization in low-ionic-strength solutions, but this effect is diminished at [KCI] = 150 mM. At low ionic strengths, micromolar concentrations of calponin induce the formation of large bundles of actin filaments, and lower concentrations of calponin quench the fluorescence of pyrene-labeled F-actin. The latter effect is related to binding of calponin to F-actin rather than to bundling of the filaments. The concentration of calponin required to bundle a fixed concentration of actin filaments increases with increasing ionic strength, as the average diameter of the bundles decreases. Millimolar concentrations of ATP, GTP or ITP are equally efficient at dispersing actin bundles to single filaments or smaller aggregates, even though a significant fraction of calponin remains bound to F-actin. Our findings show that the binding of calponin to actin is determined at least in part by electrostatic interactions, and that the polycationic nature of calponin is primarily responsible for the formation of F-actin bundles via its ability to reduce the electrostatic repulsion between the negatively charged actin filaments.

Actins↗
PubMed1997

Localization of protein regions involved in the interaction between calponin and myosin.

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard alpha-calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin.

Amino Acid Sequence↗
PubMed1997

Correlation between calponin and myosin subfragment 1 binding to F-actin and ATPase inhibition.

Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the present work we have examined the effect of recombinant chicken gizzard alpha-calponin (R alpha CaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one R alpha CaP molecule to every three to four actin monomers is sufficient for maximal inhibition of acto-S1 ATPase. At this R alpha CaP/actin ratio R alpha CaP does not interfere with S1 binding to F-actin. At higher concentrations, R alpha CaP displaces S1 from F-actin and a 1:1 R alpha CaP-actin monomer complex is formed. R alpha CaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between R alpha CaP and troponin I in their actin-binding regions.

Actins↗
PubMed1997

Serotonin syndrome associated with nefazodone and paroxetine.

A 51-year-old woman was brought to the emergency department unresponsive, hyponatremic, and diaphoretic, with muscle rigidity. Her condition improved dramatically after supportive treatment and dantrolene. The patient had received nefazodone for 6 months, but the drug had been discontinued 2 days before admission after a tapering period. One day before admission, paroxetine 20 mg/day was started. A repeat challenge with paroxetine 7 days after discontinuation of nefazodone did not result in recurrence of the patient's symptoms. We believe this to be the first report of serotonin syndrome associated with nefazodone and paroxetine.

Antidepressive Agents, Second-Generation↗
PubMed1997

Ratio of inner cell mass and trophoblastic cells in blastocysts derived from porcine 4- and 8-cell embryos and isolated blastomeres cultured in vitro in the presence or absence of protein and human leukemia inhibitory factor.

In this study we investigated effects of developmental stage at onset of individual in vitro culture on the progress of development in intact embryos and individual blastomeres derived from 4-cell and 8-cell porcine embryos (referred to as 1/4 and 1/8, respectively), the necessity of serum or BSA supplementation in culture media for embryos and blastomeres (in contrast to development in a defined medium), and the role of two concentrations of human leukemia inhibitory factor (hLIF) on development of blastomeres and embryos. More (p < 0.05) 1/4 blastomeres developed to the blastocyst stage than did 1/8 blastomeres. In the serum-supplemented medium, the percentage of inner cell mass (ICM)/total cells in 8-cell- and 1/8-derived blastocysts was higher (p < 0.05) than that in 4-cell- and 1/4-derived embryos. Development to blastocysts was similar in BSA-supplemented and defined medium as compared to that in serum-enriched medium in intact 4-cell and 8-cell embryos, and 1/4 blastomeres. More (p < 0.05) 1/8 blastomeres developed to the blastocyst stage in serum-supplemented medium than in defined medium. The high hLIF concentration (1000 IU/ml) decreased (p < 0.05) blastocyst development in 1/4 blastomeres in defined medium, but fewer blastocysts (p > 0.05) lacked an ICM (blastocyst-like vesicles) than in defined medium without hLIF. It is concluded that 1) porcine intact embryos and isolated blastomeres can be cultured individually in defined medium up to the blastocyst stage from the 4-cell stage onwards; 2) more 1/4 isolated blastomeres develop to blastocysts with more total cells but with a lower ratio of ICM to total cells than blastocysts derived from 1/8 blastomeres; 3) the effects of hLIF are dependent on proteins present in the culture medium and on the embryonic stage; and 4) in defined medium, high concentrations of hLIF are inhibitory to blastocyst formation, but fewer blastocyst-like vesicles are formed. The defined culture system employed in this study allows examination of the effects of growth factors or cytokines in porcine early embryonic development.

Animals↗
PubMed1997

[Simultaneous coronary artery bypass grafting with other cardiovascular surgical procedures].

To improve the effect and reduce the mortality of the simultaneous coronary artery bypass grafting (CABG) with other cardiovascular surgical procedures, from Nov, 1984 to July, 1996, 51 patients underwent such operation. Among them 45 patients had valvular heart diseases, 4 postinfarction ventricular septal defect and ventricular aneurysm, and 1 myxoma of left atrium and abdominal aortal aneurysm. The operative mortality was 5.85% (3/51), and 3 patients died. Cardiovascular surgical patients of over 50 years or with angina pectorsi and ECG confirmed myocardiac ischemia should undergo coronary angiography routinely. If main coronary artery branches stenosis occupied over 50%, CABG must be performed. During the operation revasculariztion should be made as full as possible to enhance myocardiac protection and reduce the ascending aortic cross-clamping time.

Adolescent↗
PubMed1996

Host range restriction of parainfluenza virus growth occurs at the level of virus genome replication.

To illuminate the molecular basis for host range restriction of parainfluenza virus replication, we have examined the types of virus macromolecules produced during abortive infection of nonpermissive MDBK cells with human parainfluenza virus type 1 (hPIV1). While these cells do not support production of hPIV1 virus, they can be infected by hPIV1 as evidenced by accumulation of intracellular viral NP and HN proteins. HPIV1 is also able to drive transcription of a synthetic analog of Sendai virus (SV) genome RNA transfected into virus-infected MDBK cells. In contrast to transcription, hPIV1 genome replication does not occur in MDBK cells. Intracellular full-length genome RNA was detected only in trace amounts 2 days after infection, and was undetectable 4 days after infection. Full-length antigenome (+) sense RNA was not detectable. Nucleocapsid complexes failed to accumulate in the cytoplasm of nonpermissive cells, and no detectable nucleocapsids were released into the medium as virus particles. The data indicate that defective vRNA synthesis and/or nucleocapsid formation is responsible for the inability of hPIV1 to grow in MDBK cells. Our data also show that hPIV1 is capable of providing all helper functions for packaging SV synthetic genome analogs into infectious particles, but these SV-specific RNAs encapsidated with hPIV1 proteins are in turn not replicated by SV proteins. These results suggest that functional protein-protein interactions between parainfluenza virus strains have more stringent requirements than do protein-RNA interactions.

Animals↗