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Chien Ho

Publications and source records attributed to Chien Ho.

At least 37 records · Page 2Linked to original sources
PubMed2004

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling.

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.

Absorption↗
PubMed2004

Side-chain assignments of methyl-containing residues in a uniformly 13C-labeled hemoglobin in the carbonmonoxy form.

Sequence-specific assignment of the methyl groups in large proteins can be obtained from an MQ-(H)CC(m)H(m)-TOCSY experiment on uniformly (13)C-labeled proteins without deuteration (Yang et al., 2004). Here the procedure is further demonstrated on a uniformly (13)C-labeled alpha-chain or beta-chain of human normal adult hemoglobin (65 kDa) in the carbonmonoxy form. In addition, a strategy is presented for assigning protons of methyl-containing residues of uniformly (13)C-labeled large proteins, on the basis of prior methyl assignments based on MQ-(H)CCH-TOCSY and H(C)C(m)H(m)-TOCSY experiments. Assignment of about 80% of the side-chain resonances of methyl-containing residues of carbonmonoxyhemoglobin has been obtained.

Carbon Isotopes↗
PubMed2004

MRI detection of tumor in mouse lung using partial liquid ventilation with a perfluorocarbon-in-water emulsion.

Transverse relaxation time (T(2*))-weighted (1)H-MRI of mouse lungs has been performed using partial liquid ventilation (PLV) with a perfluorocarbon (PFC)-in-water emulsion as a contrast modality for lung MRI. Significant sensitivity enhancement in MRI of mouse lungs has been demonstrated with the protocol. The results show that the T(2*) value in lung is approximately proportional to the infusion dose up to a dose of 5 ml/kg body weight (BW) (4.5 g PFC/kg BW) and becomes essentially constant beyond this dosage. T(2*) maps of lungs have been calculated and T(2*) in lungs is in the range of 10-35 ms with this technique, which is an order of magnitude greater than the T(2*) value of mouse lungs without using a PFC-in-water emulsion. T(2*)-weighted (1)H-MR images of mouse lungs have been obtained with good quality under our experimental conditions. We have applied this technique to detect tumors in mouse lungs. Our technique can detect small lung tumors of B16 melanoma, about 1 mm in diameter, in mice. With its significant MR sensitivity enhancement and technical simplicity, T(2*)-weighted (1)H-MRI using PLV with PFC-in-water emulsion offers a promising approach to investigate lung cancers using rodent models.

Animals↗
PubMed2004

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain.

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the alpha1beta1 and the alpha1beta2 interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the alpha1beta1 and alpha1beta2 interfaces as reflected by 1H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment alpha(28-35) is in a helical conformation, while the segment alpha(19-22) is the nonhelical AB region. The shortened B-helix conserves the interactions of alpha1beta1 interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of alpha(23-26) without perturbing its overall global conformation.

Hemoglobin A↗
PubMed2004

Normal and transplanted rat kidneys: diffusion MR imaging at 7 T.

PURPOSE: To investigate the feasibility of obtaining reproducible apparent diffusion coefficient (ADC) maps of normal rat kidneys by using respiratory-triggered spin-echo diffusion-weighted magnetic resonance (MR) imaging, to investigate the sensitivity of ADC maps in the evaluation of renal blood flow, and to use this technique to monitor acute graft rejection in transplanted rat kidneys. MATERIALS AND METHODS: Spin-echo diffusion-weighted MR imaging measurements were performed in 20 normal rats and nine rats that had undergone transplantation (six rats had received allografts; three had received isografts) at 7 T. To evaluate the effect of alteration in blood flow and water transport function, angiotensin II was infused in six normal rats and a series of spin-echo diffusion-weighted MR images was obtained at five time points. Transplanted kidneys were monitored by obtaining spin-echo diffusion-weighted MR images and gradient-echo MR images every 2 hours for 8 hours on postoperative day 4. Statistical analysis was performed with repeated-measures multivariate analysis of variance and the paired t test. RESULTS: No significant differences in ADC values were observed between right and left kidneys in all three orthogonal directions; however, a small difference was observed between the cortex and medulla. ADC values in the cephalocaudal and mediolateral directions were higher than those in the anteroposterior direction (P <.01 for all). ADC values in the cortex and medulla decreased significantly (by >35%, P <.01) during angiotensin II-induced reduction in renal blood flow. No significant signal intensity change was observed between native and transplanted kidneys on gradient-echo MR images. Allografts exhibited decreased ADC values (P <.01) and isografts exhibited similar ADC values compared with native kidneys. CONCLUSION: These findings suggest that reproducible renal ADC maps can be obtained in rats by using spin-echo diffusion-weighted MR imaging at 7 T. Spin-echo diffusion-weighted MR imaging may have potential as a noninvasive tool for monitoring early graft rejection after kidney transplantation.

Angiotensin II↗
PubMed2004

A non-invasive approach to detecting organ rejection by MRI: monitoring the accumulation of immune cells at the transplanted organ.

Organ transplantation is the generally preferred medical procedure of treatment for patients with end-stage organ failure. The immunological reaction of rejection is a major cause of functional failure in transplant patients. The current "gold standard" for detecting or confirming graft rejection following solid organ transplantation requires biopsy samples in order to detect immune cell (e.g., T-cells, macrophages, etc.) infiltration into the graft and other pathological changes. This procedure is not only invasive, having associated risks, but is also prone to sampling errors that can yield false negative results. To circumvent the need for biopsies, we are developing magnetic resonance imaging (MRI) techniques to monitor the accumulation of immune cells at the transplanted organ as a means to detect graft rejection. By labeling immune cells with an MRI contrast agent, dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles, we can monitor the accumulation of these labeled immune cells at the rejecting graft as a non-invasive method to detect graft rejection. Cells can be labeled ex vivo and then infused into the animal, or MRI contrast agents can be introduced directly into the animal in vivo. Our results show excellent correlation among the MRI signal intensity due to the USPIO-labeled macrophages at the rejecting graft, immuno-staining for macrophages, histo-pathology for graft rejection, and the iron staining of tissue samples. In this article, we shall give a summary of our progress from detecting single immune cells in vitro to monitoring the accumulation of immune cells in vivo at the transplanted kidneys, hearts, and lungs in our rat models for organ transplantation by MRI.

Animals↗
PubMed2003

Superparamagnetic iron oxide particles transactivator protein-fluorescein isothiocyanate particle labeling for in vivo magnetic resonance imaging detection of cell migration: uptake and durability.

Conjugation of dextran-coated superparamagnetic iron oxide (SPIO) particles with transactivator protein (Tat)-peptide and fluorescein isothiocyanate (FITC) allows cells to readily uptake SPIO particles. This makes possible high-resolution, real-time imaging of these cells by magnetic resonance imaging (MRI). First, we need to understand how various subpopulations take up and maintain SPIO particles. In this report, we have focused on differences in T cells, B cells, and macrophages with respect to cross-linked (CL)-SPIO Tat-FITC particle uptake over 72 hours. We have found that cells quickly take up the particles and that the bead loss that does occur is not related to cell death or apoptosis. In contrast with reports in the literature, we have observed migration of the Tat-peptide conjugates primarily to the cytoplasm rather than the nucleus.

Animals↗
PubMed2003

Quaternary structure of hemoglobin in solution.

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of (15)N-(1)H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.

Carboxyhemoglobin↗
PubMed2003

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation.

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.

Adult↗
PubMed2003

Improving spatiotemporal resolution of USPIO-enhanced dynamic imaging of rat kidneys.

This paper addresses the problem of enhancing spatiotemporal resolution of ultra-small superparamagnetic iron oxide (USPIO)-enhanced dynamic MRI of rat kidneys. To alleviate the limited resolution problem of conventional full-scan Fourier imaging methods, we use a generalized series-based imaging scheme to reduce coverage of kappa-space. Experimental results demonstrate that the generalized series imaging method with basis functions constructed using two references (pre- and post-contrast) can reduce the number of phase encodings measured during the dynamic contrast wash-in process by a factor of 4 with a negligible or minimal loss of image quality. The method is expected to make 3D studies possible using USPIO-enhanced dynamic imaging of rat kidneys, and prove valuable for early detection of renal rejection after kidney transplantation.

Animals↗
PubMed2003

Probing the conformation of hemoglobin presbyterian in the R-state.

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the alpha1beta2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(beta). In short, Lys-108(beta) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.

Allosteric Regulation↗
PubMed2003

Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly-->Cys.

We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.

Amino Acid Substitution↗
PubMed2002

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine.

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.

2,3-Diphosphoglycerate↗