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Chien Ho

Publications and source records attributed to Chien Ho.

14 recordsLinked to original sources
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↗
PubMed2002

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins.

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.

Amino Acid Substitution↗
PubMed2002

Recombinant hemoglobins with low oxygen affinity and high cooperativity.

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.

Adult↗
PubMed2002

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces.

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.

Amino Acid Substitution↗
PubMed2002

MRI of lungs using partial liquid ventilation with water-in-perfluorocarbon emulsions.

A novel (1)H-MRI contrast modality for rat lungs has been developed using water-in-perfluorocarbon (PFC) emulsions for partial liquid ventilation (PLV). The feasibility of the new ventilation protocol for (1)H-MRI studies of lungs has been demonstrated. (1)H-MR images of lungs have been obtained with sensitivity and spatial resolution higher than those of the (19)F-MRI of lungs previously reported. Diffusion-weighted MRI measurements of lungs showed that the results obtained are related to the pulmonary architecture and functional properties of lungs. Although the methodology needs further improvement and evaluation, it appears to have great potential in a wide range of new applications in the field of lung MRI, such as in vivo detection of lung cancer, emphysema, and allograft rejection following lung transplantation. The ability of this technique to achieve high-quality MR images of lungs, together with its technical simplicity, stability, and low cost, makes this method a promising imaging technique for the lungs.

Animals↗
PubMed2002

Short planar gradient coils for MR microscopy using concentric return paths.

The aim of this work is to design a set of gradient coils with an optimal geometry for magnetic resonance microscopy studies. Designs for a three-axis gradient coil system particularly suited for studies with small radiofrequency coils are presented. The novel geometry involves a planar section with concentric return paths to keep the coil short. Reduction of the external field has been attempted by varying the positions of the return paths using a simulated annealing algorithm. A biplanar version of x- and z-directed prototype gradients was built and tested. A 2D-MR image of a grid phantom has been obtained on a 7-T MR instrument to demonstrate the theory. A three-axis set used as a surface gradient set has also been built and used to obtain high-resolution MR images.

Equipment Design↗
PubMed2002

In vivo detection of acute rat renal allograft rejection by MRI with USPIO particles.

BACKGROUND: Magnetic resonance imaging (MRI) for non-invasively detecting renal rejection was developed by monitoring the accumulation of macrophages labeled with dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles at the rat renal allografts during acute rejection. METHODS: Five groups of male rats with DA-->BN renal allografts and one group with BN-->BN renal isografts were investigated by MRI before, immediately after, and 24 hr after intravenous infusion with different doses of USPIO particles. All infusions were done on post-operative day 4. MRI experiments were carried out in a 4.7-Tesla instrument using a gradient echo sequence. RESULTS: MR signal intensity (MRSI) of the cortex was found to decrease with higher dosages of USPIO particles. In the absence of USPIO infusion, a decrease in MRSI was seen in the medulla region, presumably due to hemorrhage associated with renal graft rejection, while no significant change was observed in the cortex. The optimal dose of USPIO particles for visualizing rejection-associated changes in our rat kidney model appears to be 6 mg Fe/kg body weight. Iron staining results correlated with the MRSI data, indicating that the signal reduction in the MR images was due to the presence of iron. Immunohistochemical results indicated that USPIO particles were mostly taken up by infiltrating macrophages in the rejecting grafts. CONCLUSIONS: Our results suggest that MRI with intravenous administration of dextran-coated USPIO particles appears to be a valuable and promising tool that can be used as a non-invasive and sensitive method to detect graft rejection in renal transplantation.

Acute Disease↗
PubMed2002

Cerebral blood flow at one year after controlled cortical impact in rats: assessment by magnetic resonance imaging.

Progressive tissue loss and delayed cognitive deficits are seen in rats during the initial year after experimental traumatic brain injury (TBI). As much as 10% of parenchymal volume is lost even in the contralateral hemisphere by 1 year after controlled cortical impact (CCI) in rats. Progressive declines in cerebral blood flow (CBF) are also associated with advanced age and neurodegenerative diseases. Surprisingly, the long-term effects of TBI on CBF remain undefined. CBF was quantified by continuous arterial spin-labeled magnetic resonance imaging (MRI) and measurements of spin-lattice relaxation time in a slice through the plane of injury at 1 year after experimental TBI produced by CCI (n = 4) or sham surgery (n = 4) in rats. CBF was quantified in six regions of interest (ROIs) that were anatomically identified on the control images in each hemisphere and included a medial cortical segment (contusion-enriched, beneath the impact site, on the ipsilateral side) cortex, hippocampus, thalamus, amygdala/pyriform cortex, and hemisphere. At 1 year after injury, CBF was dramatically (96%) reduced in structures within the large cystic lesion that was seen in three of four rats and variably included cortex and hippocampus. Overall, there was an 80% reduction in CBF in the ipsilateral medial cortical segment comparing CCI and sham groups. Similarly, 52% and 67% reductions were seen in CBF in the cortical and hippocampal ROIs ipsilateral to impact (CCI vs. sham), respectively. These are regions both with marked CBF disturbances early after injury and that ultimately suffer considerable tissue loss over the 1-year interval. However, at 1 year after CCI, CBF was not different from sham in other ROIs, including ipsilateral thalamus, or either contralateral hippocampus or hemisphere. We conclude that, at 1 year after CCI, CBF is reduced in anatomic structures at or near the impact site, including injured cortex and hippocampus, and this translates into a reduction in hemispheric CBF. However, despite both significant occult tissue loss ipsilateral and contralateral to the injury and delayed cognitive deficits, widespread reductions in CBF are not observed. This suggests the possibility of remodeling or repackaging of the brain that preserves CBF outside of the cystic lesion.

Animals↗