PubMed Health⌕ Search

Biomedical subjects

T Tao

Publications and source records attributed to T Tao.

At least 73 records · Page 4Linked to original sources
PubMed1996

Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein.

The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.

Base Sequence↗
PubMed1996

Immunocytochemical localization of caldesmon and calponin in chicken gizzard smooth muscle.

The distribution of caldesmon and calponin in chicken gizzard smooth muscle was investigated with immunofluorescence and immunogold electron microscopy. Immunofluorescence microscopy showed that in verapamil treated (relaxed) muscles the distributions of caldesmon and myosin appeared to be uniform throughout the cytoplasm, but clearly more textured than that of actin filaments as revealed by the distribution of tropomyosin. In shortened muscles both caldesmon and myosin became segregated, in contrast to the distribution of actin, which remained uniform. The distribution of calponin was even more textured, with no similarity to those of caldesmon or myosin. Instead, considerable overlap was observed between calponin and the cytoskeletal protein desmin and, to a lesser extent, beta-actin. By immunogold electron microscopy caldesmon appeared mostly near and around myosin filaments in both relaxed and shortened muscle. Calponin, on the other hand, was found primarily at the periphery of cytoskeletal structures in the same general region as desmin, and very often adjacent to beta-actin, which is mainly in the core. These observations indicated that caldesmon and calponin are associated with different subsets of actin filaments, caldesmon with contractile actin, while calponin with cytoskeletal actin. Thus the in situ localization of caldesmon is consistent with its proposed regulatory function. Calponin, on the other hand, is unlikely to directly regulate actomyosin interactions in these cells; instead, it may function as a bridging protein between the actin and the intermediate filament networks.

Animals↗
PubMed1996

Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.

Adenosine Triphosphate↗
PubMed1996

Effects of calponin on isometric force and shortening velocity in permeabilized taenia coli smooth muscle.

Calponin, a thin filament-associated protein, inhibits actomyosin adenosinetriphosphatase in solution and has been suggested to modulate smooth muscle contractility. We used permeabilized guinea pig taenia coli smooth muscle to investigate whether calponin can modulate actin-myosin interaction in a more organized contractile system. Fibers were permeabilized with Triton X-100 and glycerol, which permit access of large macromolecules to the contractile apparatus. For contractures elicited by Ca2+ (6.6 microM + 0.1 microM calmodulin), the recombinant alpha-isoform of chicken gizzard calponin (CaP) decreased isometric force (Fo) and unloaded shortening velocity (Vus) in a dose-dependent manner; 1 microM CaP had minimal effects on force (< 10%) but reduced Vus by approximately 50% and 10 microM CaP reduced Fo to 27% of control and Vus to near zero levels. To eliminate any effects of the binding of calmodulin by CaP and consequent inhibition of myosin light chain kinase activity, we also studied fibers activated by thiophosphorylation of the myosin regulatory light chain. Fo was only moderately inhibited, remaining at approximately 75% of control in the presence of CaP (10 microM), whereas Vus was reduced to 32% of control. A similar inhibition was obtained with a mutant (CaPcys175) that retains the ability to bind to actin. CaP phosphorylated by protein kinase C and CaPcys175 mutant labeled with 1,5-IAEDANS, which bind actin poorly, were not effective inhibitors. Our results indicate that 1) CaP more strongly inhibits Vus (approximately cross-bridge cycle rate) than Fo (approximately number of activated cross bridges) and 2) the effects of CaP are related to its binding to actin. Thus the function of CaP in regulation of smooth muscle contractility may be more strongly related to its function as a modulator of velocity, as related to the "latch state," than as an "on-off" switch.

Adenosine Triphosphate↗
PubMed1996

Effects of calponin on force generation by single smooth muscle cells.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

Animals↗
PubMed1995

Proximity relationships between engineered cysteine residues in chicken skeletal myosin regulatory light chain. A resonance energy transfer study.

Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys2 and Cys155 and Cys73 and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys2 and Cys155 was measured to be 35 and 30 A in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys73 and Cys155 measured in a similar manner was 31 and 30 A in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.

Animals↗
PubMed1995

Physical characterization of calponin. A circular dichroism, analytical ultracentrifuge, and electron microscopy study.

Calponin is a thin filament-associated smooth muscle protein that has been suggested to play a role in the regulation of smooth muscle contraction. We have used circular dichroism spectroscopy, electron microscopy, and analytical ultracentrifugation to study the physical properties of recombinant chicken gizzard alpha-calponin. The alpha-helix content of alpha-calponin was estimated from its circular dichroism spectrum to be approximately 13%, alpha-Calponin melts with a single sharp transition at approximately 57 degrees C. Rotary shadowing electron micrographs of alpha-calponin reveal diverse shapes ranging from elongated rods to collapsed coils. The lengths of the rod-shaped structures are approximately 18 nm. Analytical ultracentrifugation studies found alpha-calponin to be homogeneous with a monomer molecular mass of 31.4 kDa, and a s20,w value of 2.34 S. These data could be used to model alpha-calponin as a prolate ellipsoid of revolution with an axial ratio of 6.16, a length of 16.2 nm, and a diameter of 2.6 nm. Taken together, our results indicate that calponin is a flexible, elongated molecule whose contour length is sufficient to span three actin subunits along the long pitch helix of an F-actin filament.

Animals↗
PubMed1995

HepG2/erythrocyte glucose transporter (GLUT1) gene in NIDDM: a population association study and molecular scanning in Japanese subjects.

To evaluate the role of mutations in the glucose transporter (GLUT1) gene in Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM), we first conducted a population association study using the XbaI polymorphism of the gene. A polymerase chain reaction (PCR)-based assay was developed and used for the analysis. When analysed in 91 diabetic patients and 87 non-diabetic control subjects, the distribution of the genotype frequency was significantly different between the two groups (p = 0.0025). The (-) allele was significantly associated with NIDDM (odds ratio 2.317, 95% confidence interval 1.425-3.768). To identify possible mutation(s) in the GLUT1 gene, which was in linkage disequilibrium with the (-) allele, all ten exons of the gene were analysed by PCR single-strand conformation polymorphism (SSCP) analysis in 53 diabetic patients with at least one (-) allele. Variant SSCP patterns were detected in exons 2, 4, 5, 7, 9 and 10. Sequence analysis revealed that all the variants represented silent mutations. One of the variants in exon 2, GCT (Ala15)-->GCC(Ala), created a HaeIII restriction site. This polymorphism was common in Japanese subjects with heterozygosity of 0.36 and polymorphism information content 0.29. We conclude that the structural mutation of GLUT1 is rare and not likely to be a major genetic determinant of NIDDM in Japanese subjects. The XbaI (-) allele of the GLUT1 gene appeared to be a genetic marker of NIDDM in Japanese subjects. The possibility of the presence of mutation(s) in the regulatory region of the gene or in another locus nearby could not be excluded.

Alleles↗
PubMed1995

In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embyo yield in vitro.

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P <or= 0.05) when 10% pFF and 1microg/ml FSH were present in the maturation medium. Another 878 COC were mechanically denuded and used for in vitro fertilization (IVF). Eighteen hours after IVF, 561 oocytes were fixed and stained with aceto orcein. No diffeences were found among groups at this time point. Another 317 embryos were continuously cultured to morula and blastocyst stages in medium NCSU 23. Cleavage rates differed significantly (P <or= 0.05) 48 h after IVF, and were superior (52.5% vs 32.5% vs 30.7%) when oocytes were matured in maturation medium supplemented with pFF and FSH as compared with maturation rates in TCM 199 without pFF and/or FSH. Once the embryos had cleaved, no further differences were observed in the development to morula and blastocyst stages. These data indicate that GVBD and nuclear matuation to metaphase II are enhanced by a combination of pFF and FSH and that cytoplasmatic maturation is more complete and better synchronized as indicated by the increased cleavage rates.

Journal Article↗
PubMed1995

Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg2+ in a manner similar to that of the mgtB P-type ATPase.

Salmonella typhimurium has three distinct Mg2+ transport systems: CorA, MgtA, and MgtB, each encoded by its respective gene. corA and mgtB have been previously sequenced and characterized. This report details the sequence and properties of mgtA. Like mgtB, mgtA encodes a P-type ATPase. The mgtA gene encodes a slightly smaller protein than does mgtB, with a predicted molecular mass of about 95 kDa, running at 91 kDa on protein gels, which compares with values of 101 and 102 kDa, respectively, for the MgtB protein. The deduced amino acid sequence of MgtA is only 50% identical to that of MgtB, with a further 25% conservative amino acid substitutions, surprisingly low for such otherwise functionally similar proteins from the same organism. Codon usage for each gene is normal for S. typhimurium, however, indicating that neither gene is the result of a recent acquisition from another organism. A single open reading frame at mgtA encodes MgtA, in contrast to mgtB, which is shown to be an operon encoding (5' to 3') the 22.5-kDa MgtC and the MgtB proteins. Genetic constructs were used to show that deletion of MgtC does not alter the expression or transport properties of MgtB, making the role of the companion MgtC protein unclear. (The S. typhimurium homolog of treR, which encodes a putative repressor for trehalose uptake, is encoded by a gene adjacent to mgtA, and its sequence is also reported. Finally, exteremely strong Mg(2+) regulation of the mgtA and mgtB promoters but not of the corA or treR promoters was demonstrated by cloning the appropriate DNA sequences with luxAB and measuring enhancement of light production as a function of extracellular Mg(2+) concentration. Lowering the extracellular Mg(2+) concentration from 10 mM to 1 or 10 microM elicited a transcriptional response of several thousandfold from both the mgtA and mgtB promoters.

Adenosine Triphosphatases↗
PubMed1995

Binding motifs predict major histocompatibility complex class II-restricted epitopes in the Sendai virus M protein.

Major histocompatibility complex (MHC) class I ligand motifs have been defined for a number of class I molecules and have been successfully used to identify class I-restricted cytotoxic T-cell epitopes. In contrast, the relative degeneracy of sequence motifs in naturally processed MHC class II ligands has suggested that they may be of more limited use. Here, we use a predicted I-Ab ligand motif to identify antigenic peptides in the Sendai virus Enders strain matrix (M) protein. The entire coding sequence of the M protein was derived, and seven peptide sequences that contained the predicted I-Ab motif were identified. Analysis of I-Ab-restricted M-specific T-cell hybridomas for reactivity to these synthetic peptides identified two distinct epitopes. These data demonstrate that MHC class II motifs can be valuable in predicting T-cell epitopes.

Amino Acid Sequence↗
PubMed1995

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants.

Ca2+ regulation of vertebrate-striated muscle contraction is initiated by conformational changes in the Ca(2+)-binding protein troponin C (TnC) and subsequent changes in the interaction of TnC with the inhibitory protein TnI. We have constructed mutants of rabbit skeletal muscle TnC in which natural Cys-98 was replaced by Leu, and a single Cys residue was introduced at position 12 (TnC12) or 89 (TnC89). Cys residues of mutant TnCs were derivatized with 4-maleimidobenzophenone and photocross-linked to TnI in binary TnC.TnI complexes. After digestion with CNBr or proteases, cross-linked peptides were purified and sequenced. TnC12 cross-linked at or near TnI Met-134 in a region known to be sensitive not only to occupancy of the regulatory Ca(2+)-binding sites of TnC but also to the contractile state of the thin filament. TnC89 cross-linked to TnI(108-113) in the inhibitory region. Taken together with earlier findings, these results indicate that in the TnC.TnI complex, both domains of TnC, as well as the linker region between them, make contact with the inhibitory region of TnI. Our data also indicate that the N- and C-terminal domains of TnC interact with opposite ends of the TnI inhibitory region.

Amino Acid Sequence↗
PubMed1994

A disulfide crosslink between Cys98 of troponin-C and Cys133 of troponin-I abolishes the activity of rabbit skeletal troponin.

Various thio-reactive bifunctional crosslinkers as well as 5,5'-dithiobis(2-nitrobenzoate)-mediated disulfide bond formation were used to crosslink troponin-C and troponin-I, the Ca(2+)-binding and inhibitory subunits of troponin, respectively. In all cases, substantial crosslinking was obtained when the reactions were carried out in the absence of Ca2+. No disulfide crosslinking occurred if either Cys98 of TnC, or Cys133 of TnI were blocked, indicating that these thiols are involved in the crosslinking. Troponin containing the disulfide crosslink is no longer capable of regulating actomyosin ATPase activity in a Ca(2+)-dependent manner. Our results suggest that the relative movement between the Cys98 region of TnC and the Cys133 region of TnI is required for the Ca(2+)-regulatory process in skeletal muscle.

Actomyosin↗
PubMed1994

Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells.

Calponin is a thin filament-associated protein that has been implicated in playing an auxiliary regulatory role in smooth muscle contraction. We have used immunofluorescence and digital imaging microscopy to determine the cellular distribution of calponin in single cells freshly isolated from the ferret portal vein. In resting cells calponin is distributed throughout the cytosol, associated with filamentous structures, and is excluded from the nuclear area of the cell. The ratio of surface cortex-associated calponin to cytosol-associated calponin (R) was found to be 0.639 +/- 0.021. Upon depolarization of the cell with physiological saline solution containing 96 mM K+, the distribution of calponin did not change from that of a resting cell (R = 0.678 +/- 0.025, P = 0.369). Upon stimulation with an agonist (10 microM phenylephrine) that is known to activate protein kinase C (PKC) in these cells, the cellular distribution of calponin changed from primarily cytosolic to primarily surface cortex associated (R = 1.24 +/- 0.085, P < 0.001). This agonist-induced redistribution of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of these cells. These results suggest that the physiological function of calponin may be to mediate agonist-activated contraction via a PKC-dependent pathway.

Actins↗
PubMed1994

Insulin-like effects of vanadate on rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA and protein inductions in diabetic rats.

Effects of vanadate on liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/F-2,6-P2ase) mRNA and protein inductions were examined in streptozotocin (STZ)-induced diabetic rats. In diabetic rats at one week after STZ (60 mg/kg body weight), the liver 6PF-2-K activity was decreased to 22% of the control. The enzyme protein was also decreased to 31% of the control, but the reduction in mRNA was not significant. Treatment of diabetes with vanadate (10 mg/kg BW, i.v., every 8 h), as well as insulin (10 u/kg BW, s.c., every 8 h), increased the 6PF-2-K activity and the enzyme protein content, though it was not completely restored to the control level. 68% of the control was the figure for enzyme activity and 65% of the control for protein content after 24-h of treatment. On the other hand, vanadate, like insulin, increased enzyme mRNA content to a higher level than the control (140% of the control). The present results indicate that vanadate, like insulin, modulates the liver 6PF-2-K/Fru-2,6-P2ase gene expression, and stimulated protein induction contributes to the regulation of its enzyme activity, resulting in amelioration of the deranged carbohydrate metabolism in the diabetic state.

Animals↗