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Biomedical subjects

T Tao

Publications and source records attributed to T Tao.

At least 91 records · Page 5Linked to original sources
PubMed1994

[A successful delivery three years after the onset of multiple fungal liver abscess in a patient with acute lymphoblastic leukemia].

A 25-year-old woman was diagnosed as having the multiple fungal liver abscess which developed just after the re-induction chemotherapy, 13 months after the onset of acute lymphoblastic leukemia. Although chemotherapy for leukemia was suspended, her leukemia did not relapse. The multiple fungal liver abscess was successfully treated with anti-fungal drugs for the following 12 months. Three years after the onset of the fungal abscess, she married, became pregnant and vaginally delivered a healthy female infant at full term. In this way, it was demonstrated that a normal pregnancy and delivery could be expected even in patients who had received intensive chemotherapy and had recovered from the fungal abscess.

Adult↗
PubMed1994

[Defects of candidate genes in Japanese NIDDM--glucose transporter gene(GLUT1 gene, GLUT4 gene)].

To assess the contribution of GLUT1 and GLUT4 genes to NIDDM susceptibility in Japanese population, we performed population studies using RFLP markers. We found the strong association between XbaI polymorphism at GLUT1 gene and NIDDM, but no association between KpnI polymorphism at GLUT4 gene and NIDDM. Based on these results, molecular scanning of GLUT1 gene was performed using SSCP and direct sequencing in Japanese population, to substantiate the gene defect predisposing to NDDM. Although silent mutations were found, the meaningful mutations within the coding regions were not demonstrated. Thus the positive association observed in population study may be spurious or due to the abnormalities in non-coding regions such as promoter or other regulatory elements at GLUT1 gene.

Asian People↗
PubMed1993

Interaction between calponin and smooth muscle myosin.

Calponin is a thin filament-associated protein in smooth muscle that has been shown to bind actin, tropomyosin and calmodulin, and has been implicated to play a role in regulation of smooth muscle contractility. Using a centrifugation assay we found that calponin interacts with unphosphorylated filamentous smooth muscle myosin. We found that this calponin-myosin interaction is reversed by Ca(2+)-CaM, and depends on ionic strength. At 50 mM NaCl the binding constant and the stoichiometry of this interaction were estimated to be 2 x 10(6) M-1, and 1.2-2.4 calponin per myosin, respectively. We suggest that the calponin-myosin interaction could be involved in regulation of smooth muscle contractility by anchoring myosin to actin.

Animals↗
PubMed1993

Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium.

P-type ATPases are a family of cation transport enzymes present in all species from bacteria to mammals whose members mediate membrane flux of all common biologically relevant cations. More than 50 members of this family of transporters have been sequenced; extensive structural data are available, and several members have been analyzed by site-directed mutagenesis. Nonetheless, there is no current consensus regarding their membrane topology. In this work, the Salmonella typhimurium Mg2+ transporting P-type ATPase encoded by the MgtB locus has been used as a model for P-type ATPases. Unlike other prokaryotic P-type ATPases, the MgtB protein is similar in length, amino acid sequence, and hydropathy profile to known eukaryotic P-type ATPases. The membrane topology of MgtB was analyzed by several epitope insertions in MgtB and from the activity of 35 protein fusions between MgtB and the reporter enzymes BlaM (beta-lactamase) and LacZ (beta-galactosidase). The epitope insertions within MgtB all retained function as assessed by cation uptake assays and were regulated normally by the level of Mg2+ within the growth medium. The epitope insertion and fusion protein data are completely incompatible with the numerous previously proposed models for P-type ATPases predicting 7, 8, 9, or 12 transmembrane segments. Rather, they indicate that MgtB contains 10 transmembrane segments with both amino and carboxyl termini residing within the cytosol. By extension, we suggest that all eukaryotic P-type ATPases contain 10 transmembrane segments with both termini within the cytosol.

Adenosine Triphosphatases↗
PubMed1993

The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region.

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.

Amino Acid Sequence↗
PubMed1993

The molecular switch in troponin C.

Conformational changes in troponin C (TnC) associated with Ca(2+)-induced triggering of muscle contraction are discussed in light of the model proposed by Herzberg, Moult and James (J. Biol. Chem. 261, 2638, 1986) and of our recent work on mutants of troponin C. The model involves a Ca(2+)-induced angular movement of one pair of alpha-helical segments relative to another pair of helices in the N-terminal domain. A disulfide bridge introduced into the N-terminal domain reversibly blocks the key conformational transition and the Ca(2+)-regulatory activity. Binding of troponin I (TnI) to the disulfide form of TnC is weakened owing to the blocking of its interaction with the N-terminal domain; however incorporation of the mutant into TnC-extracted myofibrils is not abolished. Introduction of a Cys residue in the C-terminal domain of TnC leads to disulfide formation between it and the indigenous Cys-98, with accompanying inhibition of regulatory activity attributable to interference with binding to TnI and, consequently, incorporation into the thin filaments. Evidence for movement of helical segments upon Ca(2+)-binding to TnC was obtained by measurements of excimer fluorescence and of resonance energy transfer with probes attached to Cys residues introduced by site-directed mutagenesis at suitable locations. Introduction of a disulfide bridge into calmodulin, another member of the super-family of Ca(2+)-binding proteins to which TnC belongs, abolishes its interaction with target enzymes. This suggests that the type of conformational change involving angular movement of helical segments that takes place in TnC is also involved in signal transmission in other Ca(2+)-dependent regulatory proteins.

Animals↗
PubMed1993

Characterization of wild type and mutant chicken gizzard alpha calponin expressed in E. coli.

Calponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction. Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, alpha and beta, the expression of the alpha-isoform being uniformly more abundant in various smooth muscle tissues [Takahashi, K. & Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288]. For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of alpha calponin. The amino acid composition and N-terminal sequence of the recombinant alpha calponin were found to be identical to those deduced from its nucleotide sequence. Recombinant alpha calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity. A mutant alpha calponin with a replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.

Actins↗
PubMed1992

Characterization of the Ca(2+)-triggered conformational transition in troponin C.

Troponin C is the Ca(2+)-binding subunit of troponin in vertebrate striated muscle. Binding of Ca2+ to troponin C is thought to induce a conformational change that triggers subsequent events in the initiation of muscle contraction. A molecular modeling study has proposed that, when Ca2+ binds to the N-terminal triggering sites, helices B and C separate from the helices D and A, thereby exposing a crucial interaction site for troponin I, the inhibitory subunit of troponin [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. In the present study the question of whether this separation actually occurs is addressed directly. A mutant rabbit skeletal troponin C containing a pair of cysteines at position 12 in helix A and position 49 in the polypeptide segment linking helices B and C was created by site-directed mutagenesis. Pyrene excimer fluorescence and resonance energy transfer studies on the labeled mutant troponin C reveal a Ca(2+)-induced increase in distance between the two cysteines. Under certain assumptions, the distance increase could be estimated from the extent of energy transfer to be approximately 13 A, in good agreement with the distance increase predicted by molecular modeling. Our results provide further experimental support for the model proposed by Herzberg et al. (above).

Animals↗
PubMed1992

Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.

Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.

Amino Acid Sequence↗
PubMed1992

Sequence and characterization of pvuIIR, the PvuII endonuclease gene, and of pvuIIC, its regulatory gene.

An open reading frame partially overlaps pvuIIR, and genetic evidence implies that this open reading frame, named pvuIIC, specifies a positive regulator of pvuIIR (T. Tao, J. C. Bourne, and R. M. Blumenthal, J. Bacteriol. 173:1367-1375, 1991). Inducible constructs of pvuIIC produced a protein of the expected size. The site of C.PvuII action appears to lie within pvuIIC itself; thus, pvuIIC may be a self-contained regulatory cassette.

Amino Acid Sequence↗
PubMed1991

Cross-linking of residue 57 in the regulatory domain of a mutant rabbit skeletal muscle troponin C to the inhibitory region of troponin I.

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. In the present study, we investigated the sites of interaction between the N-terminal regulatory domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57) that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone (BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis. The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121. Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a complex with TnI, TnC adopts a more compact conformation than in the crystal structure.

Amino Acid Sequence↗
PubMed1991

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin.

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.

Actins↗
PubMed1991

On the relationship between distance information derived from cross-linking and from resonance energy transfer, with specific reference to sites located on myosin heads.

The techniques of fluorescence resonance energy transfer (FRET) and cross-linking can provide complementary information concerning the relative separation of a pair of sites. Cross-linking experiments provide an assessment of the distance of closest approach between a pair of sites. FRET measurements, by contrast, yield information about the average distance between the pair of sites. We have taken advantage of hybrid myosins to understand the relationship between distances obtained for a pair of equivalent sites, one on each myosin head, using both FRET (steady-state and time-decay) and cross-linking techniques. The rigid cross-linker, 4-4'-dimaleimidyl-stilbene-2-2'-disulfonic acid (DMSDS), can efficiently cross-link the two myosin regulatory light-chains, each at residue Cys50 of the Mercenaria regulatory light chain (Chantler, P.D., and S. M. Bower. 1988. J. Biol. Chem. 263:938-944), indicating that these sites can come within 18 +/- 2 A of each other. In a complementary set of experiments, steady-state and time-decay measurements using fluorescence donor/acceptor pairs located at these same sites indicate transfer efficiencies of somewhat less than 20%, suggesting an average separation of greater than 50 A between sites (Chantler, P. D., and T. Tao. 1986. J. Mol. Biol. 192:87-99). Here, we present theoretical calculations which show that efficient cross-linking can be achieved readily in dynamic systems such as the heads of myosin, even though the necessary subpopulation of proximate molecules at any instant may be below the detection limits of time-decay-FRET. Therefore, cross-linking experiments can provide important ancillary information about the extent of motions within a marcomolecular system when used in conjunction with FRET.As a corollary, demonstration of extensive cross-linking does not necessarily indicate a static proximity; the mean separation distance should be ascertained by other methods such as FRET.

Binding Sites↗
PubMed1991

A family of regulatory genes associated with type II restriction-modification systems.

Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell. An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family. First, interruptions of this ORF result in a nonrestricting phenotype. Second, this ORF can restore restriction competence to such interrupted mutants in trans. Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif. A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF. All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism.

Amino Acid Sequence↗
PubMed1990

Inhibition of mutant troponin C activity by an intra-domain disulphide bond.

Triggering of contraction in striated muscles involves a conformational transition in the N-terminal domain of troponin C, the calcium-binding component of thin filaments. We have designed a mutant troponin C in which the key conformational transition and the calcium-regulatory activity are reversibly blocked by the formation of a disulphide bridge. Our results may be applicable to other proteins of the same family of calcium-binding proteins.

Animals↗
PubMed1990

Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C.

We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.

Animals↗