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C Ho

Publications and source records attributed to C Ho.

At least 91 records · Page 5Linked to original sources
PubMed1997

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed gamma-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with gamma-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Disheveled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Amino Acid Sequence↗
PubMed1997

Interaction of alcohols and anesthetics with protein kinase Calpha.

The key signal transduction enzyme protein kinase C (PKC) contains a hydrophobic binding site for alcohols and anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKCalpha results in dramatically different effects on membrane-associated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKCalpha differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKCalpha contains two distinct phorbol ester binding regions of low and high affinity for the activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). Short chain n-alkanols competed for low affinity phorbol ester binding to the enzyme, resulting in reduced enzyme activity, whereas high affinity phorbol ester binding was unaffected. Long chain n-alkanols not only competed for low affinity phorbol ester binding but also enhanced high affinity phorbol ester binding. Furthermore, long chain n-alkanols enhanced phorbol ester induced PKCalpha activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the n-alkanols alone were weak activators of the enzyme. The cellular effects of n-alkanols and general anesthetics on PKC-mediated processes will therefore depend in a complex manner on the locality of the enzyme (e.g. cytoskeletal or membrane-associated) and activator type, apart from any isoform-specific differences. Furthermore, effects mediated by interaction with the region on the enzyme possessing low affinity for phorbol esters represent a novel mechanism for the regulation of PKC activity.

Alcohols↗
PubMed1997

Automated probabilistic method for assigning backbone resonances of (13C,15N)-labeled proteins.

We present a computer algorithm for the automated assignment of polypeptide backbone and 13C beta resonances of a protein of known primary sequence. Input to the algorithm consists of cross peaks from several 3D NMR experiments: HNCA, HN(CA)CO, HN(CA)HA, HNCACB, COCAH, HCA(CO)N, HNCO, HN(CO)CA, HN(COCA)HA, and CBCA(CO)NH. Data from these experiments performed on glutamine-binding protein are analyzed statistically using Bayes' theorem to yield objective probability scoring functions for matching chemical shifts. Such scoring is used in the first state of the algorithm to combine cross peaks from the first five experiments to form intraresidue segments of chemical shifts (Ni,HiN,Ci alpha, Ci beta, Ci'), while the latter five are combined into interresidue segments (Ci alpha,Ci beta,Ci',Ni + 1,Hi + 1N). Given a tentative assignment of segments, the second stage of the procedure calculates probability scores based on the likelihood of matching the chemical shifts of each segment with (i) overlapping segments; and (ii) chemical shift distributions of the underlying amino acid type (and secondary structure, if known). This joint probability is maximized by rearranging segments using a simulated annealing program, optimized for efficiency. The automated assignment program was tested using CBCANH and CBCA(CO)NH cross peaks of the two previously assigned proteins, calmodulin and CheA. The agreement between the results of our method and the published assignments was excellent. Our algorithm was also applied to the observed cross peaks of glutamine-binding protein of Escherichia coli, yielding an assignment in excellent agreement with that obtained by time-consuming, manual methods. The chemical shift assignment procedure described here should be most useful for NMR studies of large proteins, which are now feasible with the use of pulsed-field gradients and random partial deuteration of samples.

Amino Acid Sequence↗
PubMed1997

1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli.

1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples. Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.

Amino Acid Sequence↗
PubMed1997

A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin.

Human haemoglobin has long been a paradigm for cooperative ligand binding and allostery. Through analysis of the crystal structures of deoxyhaemoglobin and liganded haemoglobin, Perutz proposed a model for cooperativity in which the bond between the proximal histidine and the protein couples haem rearrangements to protein structure rearrangements. Here we test this model by deleting the bonds between the histidine imidazole side chain and the polypeptide. This detachment method allows us to determine directly the contribution of proximal histidine coupling to cooperativity of distal ligand binding. Proximal detachment significantly increases ligand affinity, reduces cooperativity, and prevents quaternary structure switching, in accord with the Perutz model. Residual cooperativity indicates that additional haem communication pathways exist that do not involve the proximal histidine coupling mechanism.

Crystallography, X-Ray↗
PubMed1997

CBFA2, frequently rearranged in leukemia, is not responsible for a familial leukemia syndrome.

We have identified a family with an autosomal dominant platelet disorder with a predisposition for developing myeloid malignancies and have previously demonstrated linkage of this trait to chromosome 21q22.1-22.2. The nearest flanking markers, D21S1265 and D21S167, define the familial platelet disorder (FPD) critical region at a genetic distance of approximately 15.2 centimorgans and physical distance of approximately 6 megabases. This locus is of particular interest as it has previously been implicated in the pathogenesis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) through the (8;21), (3;21) and (12;21) chromosomal translocations. In each of these cases, the CBFA2 gene is rearranged. As well, there is a potential association of this locus with the hematologic abnormalities seen in Down syndrome (trisomy 21). To identify the mutant gene in this pedigree, a positional cloning strategy has been undertaken. Several candidate genes map to this locus including: CBFA2, IFNAR1, IFNAR2, CRFB4, GART, SON, KCNE1, SCL5A3 and ATP50. CBFA2, as well as IFNAR1 and CRFB4, were the focus of initial mutational analysis efforts. In this report, we exclude CBFA2 as a candidate by Northern and Southern blotting, RNase protection, single-strand conformational polymorphism (SSCP), direct sequencing and gel-shift analysis. Exons of the IFNAR1 and CRFB4 genes were also analyzed by SSCP and demonstrated no evidence of mutation. SSCP analysis identified a new polymorphism in the second exon of the CRFB4 gene and confirmed a previously described polymorphism in the fourth exon of IFNAR1. Efforts are currently underway to delimit further the FPD critical region and to analyze the other known candidate genes, as well as novel candidate genes, which map to this locus.

Core Binding Factor Alpha 2 Subunit↗
PubMed1997

Exceptional convergent evolution in a virus.

Replicate lineages of the bacteriophage phiX 174 adapted to growth at high temperature on either of two hosts exhibited high rates of identical, independent substitutions. Typically, a dozen or more substitutions accumulated in the 5.4-kilobase genome during propagation. Across the entire data set of nine lineages, 119 independent substitutions occurred at 68 nucleotide sites. Over half of these substitutions, accounting for one third of the sites, were identical with substitutions in other lineages. Some convergent substitutions were specific to the host used for phage propagation, but others occurred across both hosts. Continued adaptation of an evolved phage at high temperature, but on the other host, led to additional changes that included reversions of previous substitutions. Phylogenetic reconstruction using the complete genome sequence not only failed to recover the correct evolutionary history because of these convergent changes, but the true history was rejected as being a significantly inferior fit to the data. Replicate lineages subjected to similar environmental challenges showed similar rates of substitution and similar rates of fitness improvement across corresponding times of adaptation. Substitution rates and fitness improvements were higher during the initial period of adaptation than during a later period, except when the host was changed.

Bacteriophage phi X 174↗
PubMed1997

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Adult↗
PubMed1997

Assessment of cerebral blood flow and CO2 reactivity after controlled cortical impact by perfusion magnetic resonance imaging using arterial spin-labeling in rats.

We measured CBF and CO2 reactivity after traumatic brain injury (TBI) produced by controlled cortical impact (CCI) using magnetic resonance imaging (MRI) and spin-labeled carotid artery water protons as an endogenous tracer. Fourteen Sprague-Dawley rats divided into TBI (CCI; 4.02 +/- 0.14 m/s velocity; 2.5 mm deformation), sham, and control groups were studied 24 hours after TBI or surgery. Perfusion maps were generated during normocarbia (Paco2 30 to 40 mm Hg) and hypocarbia (PaCO2 15 to 25 mm Hg). During normocarbia, CBF was reduced within a cortical region of interest (ROI, injured versus contralateral) after TBI (200 +/- 82 versus 296 +/- 65 mL.100 g-1.min-1, P < 0.05). Within a contusion-enriched ROI, CBF was reduced after TBI (142 +/- 73 versus 280 +/- 64 mL.100 g-1.min-1, P < 0.05). Cerebral blood flow in the sham group was modestly reduced (212 +/- 112 versus 262 +/- 118 mL.100 g-1.min-1, P < 0.05). Also, TBI widened the distribution of CBF in injured and contralateral cortex. Hypocarbia reduced cortical CBF in control (48%), sham (45%), and TBI rats (48%) versus normocarbia, P < 0.05. In the contusion-enriched ROI, only controls showed a significant reduction in CBF, suggesting blunted CO2 reactivity in the sham and TBI group. CO2 reactivity was reduced in the sham (13%) and TBI (30%) groups within the cortical ROI (versus contralateral cortex). These values were increased twofold within the contusion-enriched ROI but were not statistically significant. After TBI, hypocarbia narrowed the CBF distribution in the injured cortex. We conclude that perfusion MRI using arterial spin-labeling is feasible for the serial, noninvasive measurement of CBF and CO2 reactivity in rats.

Animals↗
PubMed1996

A 19F-NMR study of the equilibrium unfolding of membrane-associated D-lactate dehydrogenase of Escherichia coli.

Partially folded protein intermediates have been observed by 19F-NMR spectroscopy during the equilibrium unfolding of the membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli by a denaturant, guanidine hydrochloride (Gdn.HCl). The results from 19F-NMR and circular dichroism spectroscopic studies suggest that the intermediates observed at low Gdn.HCl concentrations (< 3.5 M) exhibit features similar to "molten globules" that contain considerable amounts of secondary and tertiary structure. The results of 19F-NMR studies on 5F-Trp-labeled D-LDH, such as the chemical shift changes, nuclear Overhauser effect, and solvent-induced isotopic shift effect, show that different regions of D-LDH unfold nonuniformly in Gdn.HCl in the presence of lysophosphatidylcholine. The polypeptide appears to unfold in a general order from the carboxyl end to the amino end, in agreement with previous findings from our laboratory that the carboxyl-terminal region of D-LDH is largely exposed to the solvent while the amino-terminal region is buried in the protein core. The structure of the partially unfolded intermediate forms of D-LDH is stabilized in the presence of lipid-like detergents, such as lysophosphatidylcholine.

Circular Dichroism↗
PubMed1996

Roles of alpha 114 and beta 87 amino acid residues in the polymerization of hemoglobin S: implications for gene therapy.

Three novel recombinant mutants of sickle hemoglobin (Hb S, beta 6Glu-->Val) have been constructed to assess the role of proline at alpha 114 and threonine at beta 87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (beta 6Glu-->Val); r Hb S-Chiapas (beta 6Glu-->Val, alpha 114Pro-->Arg); r Hb S-D-Ibadan (beta 6Glu-->Val, beta 87Thr-->Lys); and r Hb S-Chiapas-D-Ibadan (beta 6Glu-->Val, alpha 114Pro-->Arg, beta 87Thr-->Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4 degrees C to 30 degrees C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (Csat) in 0.1 M potassium phosphate (pH 7.35) at 25 degrees C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues alpha 114Pro and beta 87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of alpha 114Pro-->Arg, which offers a novel opportunity to use an alpha-chain construct, in addition to a beta-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.

Adult↗
PubMed1996

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Animals↗
PubMed1996

Contributions of asparagine at alpha 97 to the cooperative oxygenation process of hemoglobin.

According to the X-ray crystallographic results from human deoxyhemoglobin, beta 99Asp at the alpha 1 Beta 2 interface forms hydrogen bonds with alpha 42Tyr and alpha 97Asn. To clarify the structural and functional roles of the hydrogen bond between alpha 97Asn and beta 99Asp, we have engineered a recombinant hemoglobin in which alpha 97Asn is replaced by Ala, and have investigated its oxygen-binding properties, and have used proton nuclear magnetic resonance spectroscopy to determine the structural consequences of the mutation. Recombinant Hb (alpha 97Asn-->Ala) shows a milder alteration of functional properties compared to the severely impaired beta 99 mutants of the human abnormal hemoglobins. The addition of inositol hexaphosphate, an allosteric effector, causes recovery of the functional properties of recombinant Hb (alpha 97 Asn-->Ala) almost to the level of human normal adult hemoglobin without this allosteric effector. r Hb (alpha 97 Asn-->Ala) shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 interface compared to those of human normal adult hemoglobin. The proton nuclear magnetic resonance spectrum of the deoxy form of this recombinant hemoglobin shows the existence of an altered hydrogen bond which is believed to be between alpha 42Tyr and beta 99Asp at the alpha 1 beta 2 interface. Thus, the present results suggest that the intersubunit hydrogen bond between alpha 97 Asn and beta 99Asp at the alpha 1 beta 2 interface is not as crucial as the one between alpha 42Tyr and beta 99Asp in the deoxy quaternary structure. Preliminary molecular dynamics simulations have been used to calculate the contributions of specific interactions of several amino acid residues in r Hb (alpha 97Asn-->Ala) to the free energy of cooperativity of this recombinant hemoglobin. The results of these calculations are consistent with the experimental results.

Adult↗
PubMed1996

Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities.

Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.

Animals↗
PubMed1996

Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

The effect of variation of the degree of cis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKC alpha and epsilon were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKC alpha. By contrast, increasing the unsaturation of PS decreased the activity of PKC alpha, and to a lesser extent PKC epsilon. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

Animals↗
PubMed1996

Clinical experience of medullary thyroid carcinoma in Chang Gung Memorial Hospital.

Total 19 cases of medullary thyroid carcinoma (MTC) diagnosed at Chang Gung Memorial Hospital from Jan. 1979 to Dec. 1994 were studied retrospectively. All of them were pathologically proved. All cases except one were thought as sporadic MTC. The gender distribution showed a female preference with female : male (F:M) ratio of 3.8:1. The mean age at diagnosis was 44.9 +/- 13.7 years. The most common clinical presentation was thyroid mass in all (100%). The staging of MTC in our cases demonstrated: 12 (63%) stage I, 1 (5%) stage II, 3 (16%) stage III, and 3 (16%) stage IV. Thyroid ultrasonography with fine-needle aspiration cytology (FNAC) were performed in 15 patients. Eleven cases were diagnosed as malignancy, including 5 cases diagnosed as MTC preoperatively. Neck nodal metastases were noted in 7 cases and all occurred in tumors larger than 3 cm. All cases received operation according to clinical stage. Six of 7 patients who had preoperative calcitonin data showed abnormal high values. After operation, there were 8 patients with persistent hypercalcitoninemia. Of these, 5 received 131I therapy which induced undetectable calcitonin level in 2 stage I patients. Three cases received external beam radiotherapy but all failed. During a period of 0.5 to 9.4 years follow-up (mean +/- SE: 3.6 +/- 0.7 years), 3 patients died of pulmonary infection, 1 lost follow-up and 15 patients are alive and well. In conclusion, the preliminary data suggests that the medullary thyroid carcinoma in Taiwan has following characteristics: (1) gender distribution showed a female preference; (2) accuracy of preoperative FNAC was 42%; (3) most of the diagnoses of MTC were made postoperatively.

Adult↗