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H C Cheung

Publications and source records attributed to H C Cheung.

At least 73 records · Page 4Linked to original sources
PubMed1987

Interactions of troponin subunits: free energy of binary and ternary complexes.

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT.TnI [TnC.(TnT.TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT.TnI. The free energies were -9.0 kcal.mol-1 for TnC.TnT, -9.2 kcal.mol-1 for TnT.TnI, and -8.7 kcal.mol-1 for TnC.(TnT.TnI). In the presence of Mg2+ the free energies of TnC.TnT and TnC.(TnT.TnI) were -10.3 and -10.9 kcal.mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal.mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT.TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal.mol-1, -18.9 kcal.mol-1, and -21.6 kcal.mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C.

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.

Actomyosin↗
PubMed1987

Local recurrences after subtotal esophagectomy for squamous cell carcinoma.

From July 1982 to June 1985, 100 patients with squamous cell carcinoma of the thoracic esophagus had esophageal resection and reconstruction using an abdominal and right thoracotomy approach (Lewis-Tanner operation). Five patients died within 30 days. The remaining 95 patients were studied prospectively for evidence of local recurrences. It was found that anastomotic recurrences occurred in eight patients, and mediastinal recurrences involving the intrathoracic stomach occurred in seven patients over a mean follow-up period of 13 months. The total local recurrence rate was 16% (15 of 95 patients). The incidence of anastomotic recurrence was shown to be related only to the length of the proximal resection margin and not related to tumor differentiation or lymph node metastases. A proximal resection margin of less than 5 cm measured at operation had a 20% risk of developing an anastomotic recurrence, and a margin of between 5 to 10 cm had an 8% risk. Mediastinal recurrences that encroached on the intrathoracic stomach were found to be related more to the extent of lateral spread of the primary tumor in the mediastinum than to the length of the resection margins. Postoperative radiotherapy in patients with palliative resections decreased the incidence of local recurrences. To reduce the incidence and consequences of local recurrence after esophagectomy, it is suggested that in patients with tumors in the upper thorax, a more complete esophagectomy is warranted, postoperative radiotherapy should be given to patients with short resection margins, and in patients with extensive mediastinal spread, use of the retrosternal route for reconstruction is preferred.

Carcinoma, Squamous Cell↗
PubMed1987

Is pyloroplasty necessary in esophageal replacement by stomach? A prospective, randomized controlled trial.

A randomized, prospective, controlled trial compared the use of pyloroplasty (study group) with nonuse of the procedure (control group) in the treatment of 72 patients with carcinoma of the thoracic esophagus. In these patients, transthoracic esophagectomy was performed, and the whole stomach was used for reconstruction in the right side of the chest. No complications due to the pyloroplasty developed in any of the patients who underwent this procedure. Gastric emptying tests that were performed at the sixth postoperative month in 37 patients demonstrated a prolongation of emptying time in the control group (p less than 0.01). The patients' symptoms, however, did not correlate well with gastric emptying time. Follow-up assessment showed that a higher proportion of patients who did not undergo pyloroplasty suffered from symptoms of incomplete emptying while eating, but the difference was not statistically significant. Moreover, the proportion of patients who were free of postoperative symptoms gradually increased in both groups and reached 100% in the study group and 89% in the control group at 2 years. In the control group, two of the three patients with prolonged gastric emptying time complained of persistent regurgitation and distending discomfort after meals (18 and 24 months after surgery). They were the only two patients who might have benefited from a drainage procedure at the initial operation. Since there apparently were no reliable operative criteria by which to identify patients who might require drainage, and since the risk associated with pyloroplasty was negligible, it would seem reasonable to perform a pyloroplasty on every patient in whom the whole stomach was used for reconstruction after esophagectomy. This procedure would benefit the few patients who might manifest symptomatic gastric stasis.

Adult↗
PubMed1986

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin.

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Proximity relationship in the binary complex formed between troponin I and troponin C.

We have determined six molecular distances among four sites in the binary complex formed between troponin C (TnC) and troponin I (TnI) by fluorescence resonance energy transfer between donor and acceptor probes that were either an intrinsic fluorophore (Trp158 of TnI) or extrinsic probes attached to the sites. The three extrinsic probes were dansylaziridine (DNZ), N'-(iodoacetyl)-N'-(8-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 5-(iodoacetamido)eosin (IAE). The four fluorophores provided four donor-acceptor pairs: DNZ----IAE, Trp----IAEDANS, IAEDANS----IAE, and Trp----DNZ. They allowed determinations of separations between specific sites from measurements of energy transfer from (1) Met25 (DNZ) to Cys98 (IAE) in TnC, (2) Trp158 to Cys133 (IAEDANS) in TnI, (3) Cys98 (IAEDANS) of TnC to Cys133(IAE) of TnI, (4) Trp158 of TnI to Cys98(IAEDANS) of TnC, and (6) Met25(DNZ) of TnC to Cys133(IAE) of TnI. Distance (1) in TnC was little affected when the isolated protein was complexed with TnI, whereas distance (2) in TnI increased by 6A (29%) when TnI was incorporated into the binary complex. In the presence of EGTA, the six donor-acceptor separations (R) in the complex were in the range 28 to 57 A based on kappa 2 = 2/3. Mg2+ had only small effects on R, but Ca2+ induced substantial increases or decreases of R in five of the six distances. These changes were not accompanied by significant changes in the axial depolarization of the fluorophores. The results indicate global structural perturbations of regions of the two proteins in the complex by Ca2+ binding to the TnC, and suggest that large-scale movements of domains of troponin subunits may be the initial molecular events that occur in the transmission of the Ca2+ signal in the regulation of contraction by calcium.

Adenosine Triphosphatases↗
PubMed1986

Probe of the (Ca2+ + Mg2+)-ATPase in erythrocyte membranes of cystic fibrosis patients.

There are several conflicting reports regarding a defective (Ca2+ + Mg2+)-ATPase in tissues from cystic fibrosis (CF) patients. The work presented in this paper represents an independent assessment of (Ca2+ + Mg2+)-ATPase function in CF and, at the same time, provides a somewhat more detailed analysis of the enzyme from CF patients than has previously existed. We found no significant differences in either the Km value for Ca2+ or the Vm value of the (Ca2+ + Mg2+)-ATPase in membrane preparations from CF patients and control subjects when the red cell membranes were prepared by methods which utilize Tris-glycylglycine-Mg2+ buffers. In contrast, the Vm value of the (Ca2+ + Mg2+)-ATPase in the preparations from CF patients was found to be lower than that from control subjects when the membranes were prepared by a series of washes with EDTA-containing buffers (i.e., 1-10 mmol/1 EDTA). The EDTA treatment, however, did not produce any significant difference in the Km between the two groups. The fluorescent ATP analogue, trinitrocyclohexadienylidine-ATP, appeared to interact with erythrocyte ghosts as evidenced from an enhancement of its fluorescence. This enhancement was greater in control preparations than in samples from CF patients. In addition, the kinetic profiles, with respect to ATP, were quite different between the two enzyme populations. The overall results suggest that the lower rate of ATP hydrolysis observed with membranes from CF patients may reflect an impaired utilization of the substrate by the (Ca2+ + Mg2+)-ATPase from these individuals.

Adenosine Triphosphate↗
PubMed1986

23Na-NMR studies of Na+ interaction with human red cell membranes from normotensives and hypertensives.

Na+ interaction with unsealed human red cell ghosts has been studied by 23Na-NMR relaxation rate (R1) measurements. Data on a total of nine subjects including seven volunteer normotensives (NBP) and two untreated hypertensives (HBP) are presented. Qualitative treatment of the data gives information on the dynamic behavior of Na+ undergoing fast exchange between the free and bound states. The excess longitudinal relaxation rate (delta R)-1 plotted against total [Na+], known as the James-Noggle plot, exhibits different behavior for NBP and HBP ghosts, with a relatively low binding constant of approx. 100 M-1 for HBP (p less than 0.025) compared to a high constant of 500-1000 M-1 for NBP. To associate our NMR data with membrane-bound (Na+ + K+)-ATPase, 23Na relaxation rates were measured in the presence of 5 mM ouabain. James-Noggle plots constructed for ouabain-sensitive excess relaxation rates show the binding for NBP to be even high affinity (greater than 10(3) M-1) but low capacity. These data may suggest that for a given amount of intracellular Na+, the binding affinity could determine the distribution of Na+ between the membrane and cytoplasm, and that the (Na+ + K+)-ATPase which is primarily responsible for the Na+ affinity might assume an abnormal transport mechanism in HBP membranes.

Adolescent↗
PubMed1986

Shrinkage of the esophagus after resection for carcinoma.

The purpose of this study was to document and quantitate the degree of shrinkage of resection margins of the esophagus following extirpation for carcinoma. Measurements were made at operation before the esophagus was removed (in-situ), when the removed specimen had been stretched maximally (stretched), with the specimen lying free (contracted), and after fixation (fixed). After resection, there was substantial shrinkage of the margins, and the upper margins underwent a greater degree of shrinkage than the lower margins, with the tumor changing little in length. In the contracted state before fixation, the upper and lower margins were reduced to 44% and 54% of their in-situ lengths, respectively; after fixation, they were 32% and 39%, respectively. Even after maximal stretching, only 73% of the upper and 89% of the lower in-situ resection margins were restored. The overall shrinkage for the whole specimen after fixation was 50%. It is concluded that there is considerable shrinkage of the resection margins in the esophagus from the time of operation to fixation, and this accounts for the discrepancy claimed by surgeons and pathologists regarding the length of the margins. This finding has relevance in the extrapolation of surgical resection margins from pathological specimens.

Carcinoma↗
PubMed1985

An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry.

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by Förster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl] amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.

Adenosine Diphosphate↗
PubMed1985

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.

Animals↗
PubMed1984

Distance between nucleotide site and cysteine-373 of G-actin by resonance energy transfer measurements.

The distance between the nucleotide site and the reactive cysteine-373 of G-actin was determined from resonance energy transfer measurements by using 1,N6-ethenoadenosine triphosphate (epsilon ATP) as the donor and 4-[N-(iodoacetoxy)ethyl N methyl]amino 7 nitrobenz 2 oxa 1,3 diazole covalently attached to the sulphydryl group as acceptor. The quenching of the lifetime of bound donor in the presence of attached acceptor arose predominantly from transfer of excitation energy. The polarization spectrum of free epilson ATP in glycerol revealed that the minimum value of its fundamental anisotropy is 0.32 at 340 nm, indicating that the maximum value of the angle between the absorption and emission dipoles of the ethenoadenosine moiety is 21 degrees. The polarization result indicates that the bound nucleotide is depolarized and has considerable motional freedom. This motion is restricted and unlikely to be rapid or isotropic during the time interval of energy transfer. The attached acceptor is highly immobile, however. The range of the donor-acceptor distance is 24-45 A. This range was not affected by polymerization. In the absence of independent structural information it is not possible to assign a single value to the donor-acceptor separation.

Actins↗
PubMed1983

Synthetic peptide analogs of skeletal troponin C: fluorescence studies of analogs of the low-affinity calcium-binding site II.

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca2+-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca2+-specific low affinity binding site II (residues 63-74) but with two amino acid substitutions. In one analog, Phe-72 was replaced by tyrosine. In the other Gly-66 was substituted by serine and Phe-72 by tyrosine. The intrinsic fluorescence of the peptides was enhanced upon addition of Tb3+ or large excess of Ca2+. From the enhancement of Tb3+ emission association constants in the range (2-3) X 10(5) M-1 and a binding stoichiometry of 1 were determined for Tb3+ binding to the peptides. Large excess of Ca2+ displaced Tb3+ from the Tb3+-peptide complexes and from these results apparent stability constants of 500-700 M-1 were deduced for Ca2+ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La3+ induced considerable perturbation of the amide proton resonances of several residues, including the aspartate at position 3, the tyrosine at position 10, and the two glutamates at the C-terminus. The results suggest involvement of these residues in cation coordination.

Amino Acids↗
PubMed1982

Interferon inhibits Sendai virus-induced cell fusion: an effect on cell membrane fluidity.

Interferon can affect several cellular functions, in addition to its antiviral activity. We report here that pretreatment of human cells with homologous interferon significantly inhibits cell fusion induced by Sendai virus and that this refractory state is accompanied by a decrease in cell plasma membrane fluidity. Multinucleate cell formation induced by beta-propiolactone-inactivated Sendai virus in human fibroblast cells (a system in which fusion results from an interaction of the viral glycoprotein with the cell membrane) was inhibited by more than 90% after addition of human interferon for 18-24 hr. This inhibition could be neutralized by antiserum to interferon. Furthermore, inhibitor studies with cycloheximide and actinomycin D clearly indicated that synthesis of protein and RNA is necessary to establish the resistant state. To determine whether the inhibition of Sendai virus-induced cell fusion resulted from interferon-induced changes at the cell plasma membrane, experiments were carried out using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene, which is capable of sensing molecular motions in the hydrocarbon core of the bilayer structure. A significant decrease in the membrane fluidity of interferon-treated cells was observed. It is likely, therefore, that the inhibitory effect on Sendai virus-induced cell fusion observed in interferon-treated cells results from an increased rigidity of the target cell membrane.

Cell Fusion↗
PubMed1982

The treatment of thoracolumbar spinal injuries with paresis by conservative versus surgical methods.

18 patients with cord or root involvement in thoracolumbar injuries underwent posterior spinal surgery, including 11 Harrington instrumentations. 11 similar patients were treated conservatively. Surgery offered no definite advantage for neural recovery, nor did it curtail the duration of hospitalisation. However, it significantly decreased post-traumatic kyphotic deformity and improved the vertebral alignment. Morbidity and implant failure were noted in 27% of the operated cases. It would appear that operative stabilisation of the thoracolumbar injuries of the spine is indicated in selected cases where there is a risk of significant post-traumatic kyphotic deformity with gross vertebral body displacement.

Adult↗