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C Ho

Publications and source records attributed to C Ho.

At least 127 records · Page 7Linked to original sources
PubMed1994

Crystals of glutamine-binding protein in various conformational states.

Crystals of glutamine-binding protein (GlnBP) in various conformational states have been obtained. Crystals of the ligand-free "open" state (denoted form B) have unit cell dimensions a = 86.3 A, b = 86.3 A, c = 81.5 A, alpha = beta = gamma = 90 degrees and diffract to about 2.3 A resolution. An analysis of the intensity data using an Requiv plot indicates that the crystal system is orthorhombic, space group P2(1)2(1)2(1). Crystals of the ligand-bound "open" state (form B*) are obtained by soaking form B crystals with glutamine (Gln) and diffract to about 1.9 A. Crystals of the GlnBP-Gln complex in a ligand-bound "closed" state (form C) belong to space group P2(1)2(1)2(1) with a = 62.0 A, b = 65.7 A and c = 121.8 A and diffract to about 2.3 A. Crystals of a selenomethionyl GlnBP (form B') are isomorphous to form B crystals and diffract to about 2.1 A resolution.

Carrier Proteins↗
PubMed1994

Different sensitivities of native and oxidized forms of Na+/K(+)-ATPase to intracellular proteinases.

Inactivation of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been implicated in ischemia-reperfusion injury to heart and other organs. Because oxidation of many proteins makes them more susceptible to degradation by intracellular proteinases, we studied the effects of several such proteinases on native and H2O2-oxidized preparations of Na+/K(+)-ATPase from canine kidney (containing alpha 1 isoform of the catalytic subunit) and rat axolemma (containing alpha 2 and alpha 3 isoforms). Lysosomal cathepsin D degraded the native and the oxidized preparations at acid pH, but it was significantly more effective against the oxidized forms. m-Calpain had little or no effect on the native Na+/K(+)-ATPase preparations, but it digested the oxidized alpha-subunits of the axolemma and the kidney enzymes. mu-Calpain's effects were similar to those of m-calpain. Multi-catalytic proteinase which is known to degrade a large number of oxidized proteins, did not affect the native or the oxidized forms of Na+/K(+)-ATPase. The findings suggest that (a) during oxidative stress there may be accelerated degradation of the oxidatively damaged Na+/K(+)-ATPase, either through internalization and transport to lysosomes, or by the action of calpains at the membrane; and (b) those isoforms of the enzyme that are more sensitive to oxidants are more susceptible to degradation by the above processes.

Animals↗
PubMed1994

The modulation of protein kinase C activity by membrane lipid bilayer structure.

The hypothesis that protein kinase C (PKC) activity is sensitive to phospholipid head group interactions was tested using lipid bilayers of defined composition with PKC purified from rat brain. The head group interactions were modulated by varying phosphatidylcholine cis-unsaturation, vesicle curvature, and by the addition of phosphatidylethanolamine and cholesterol. With unilamellar vesicles (including 20 mol% brain phosphatidylserine), increased phosphatidylcholine unsaturation potentiated basal and phorbol ester stimulated PKC activity. By contrast, in the presence of phosphatidylethanolamine, the activity decreased with increasing phosphatidylcholine unsaturation. Weakening phospholipid head group interactions spaces the head group region and increases interstitial water, and this effect was assessed from its effect on the fluorescence intensity of the phospholipid-labeled fluorophore 1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)aminohexanoylphosphat idylcholin e (C6-NBD-PC). When the PKC activities with vesicles of varying phosphatidylcholine unsaturation, with and without phosphatidylethanolamine, were plotted as a function of the fluorescence intensity of C6-NBD-PC-labeled vesicles, a biphasic profile was obtained, which had an optimum value of intensity, relating to head group spacing, that corresponded to a maximal enzyme activity. A similar biphasic curve was also found when PKC activities were plotted as a function of published bilayer intrinsic curvature x-ray diffraction data, a parameter closely related to head group spacing. By contrast, no simple relationship was evident between PKC activity and 1,6-diphenyl-1,3,5-hexatriene anisotropy, taken as a measure of lipid order or fluidity. Therefore, increasing the level of phosphatidylcholine unsaturation, phosphatidylethanolamine, or cholesterol either potentiates or attenuates PKC activity, dependent on whether the initial condition is above or below its optimum.

Animals↗
PubMed1994

Chronic ethanol intoxication induces adaptive changes at the membrane protein/lipid interface.

Modifications were found to occur at the membrane protein/lipid interface of liver microsomes in animals that had been subjected to chronic ethanol ingestion. The effects were revealed by probing this region with 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl) phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, DPH-PC). In intact membranes, it was found that the decay of the excited state was heterogeneous, this being modeled by fitting the data to a fluorescence lifetime distribution. The full-width of the distribution at half-maximum, which relates to the degree of excited state environmental heterogeneity, increased for each fluorophore, as a result of chronic ethanol treatment. For TMA-DPH and DPH the excited state heterogeneity could have arisen from, (i) the protein/lipid interface and (ii) varied degrees of water penetration into the lipid, due to the ability of these fluorophores to sample along the bilayer normal. By contrast, the DPH in DPH-PC, due to its tethering, was only able to sample the heterogeneity at the protein/lipid interface, as confirmed by a homogeneous decay in vesicles of microsomal lipid extracts. The increased degree of DPH-PC fluorescence decay heterogeneity in microsomes from chronic ethanol-treated animals as compared to controls, was found to persist in vesicles of extracted lipids, when apocytochrome C was included in the vesicle preparations as a model protein. This effectively eliminated a protein modification from being responsible and indicated that a chronic-ethanol induced alteration in the lipids was being expressed in the form of a physico-chemical modification at the protein/lipid interface. The degree of DPH-PC environmental heterogeneity was also directly increased by ethanol, however, membranes from chronic ethanol-treated animals were resistant to this effect, showing that the phenomenon of 'membrane tolerance' extends to the membrane protein/lipid interface.

Alcoholism↗
PubMed1994

Lactate mapping with full sensitivity by spin-filtered NMR imaging.

The mechanism of the fourfold signal loss during the cyclic polarization-transfer (CYCLPOT) sequence for lactate filtering is analyzed using the spin-product-operator formalism. Based on the results of the analysis, a new sequence, RECYCLPOT (refocused CYCLPOT), is proposed and experimentally tested for lactate filtering with full sensitivity and specificity. Lactate mapping is performed by spin filtering using the RECYCLPOT sequence prior to imaging by spatial encoding.

Animals↗
PubMed1994

A complex bilateral polysyndactyly disease locus maps to chromosome 7q36.

We demonstrated that the gene responsible for a congenital limb deformity (polysyndactyly) maps to chromosome 7q36 in a large family. Pre- and postaxial anomalies of the extremities are inherited in this family as an autosomal dominant trait. The disease locus is closely linked to D7S550 (maximum lod score = 6.85, theta = 0). This region is homologous to a segment of mouse chromosome 5, where the mutations hammer toe (HM) and hemimelic extra toes (HX) have been mapped. These data suggest that human chromosome 7q36 and the homologous region of mouse chromosome 5 contain genes involved in limb pattern formation.

Base Sequence↗
PubMed1994

Structure and self assembly of a retrovirus (FeLV) proline rich neutralization domain.

The 60 amino acid proline-rich neutralization domain of the external surface unit glycoprotein of feline leukemia virus was chemically synthesized in total and in fragments. We examined the ability of these retroviral peptides to form ordered conformations using 1H-NMR, circular dichroism spectroscopy, and intrinsic viscosity measurements. One dimensional nuclear magnetic resonance spectroscopy revealed that the 60 amino acid peptide could form a stable, folded structure that was long-lived, as shown by the ability to protect amide-protons in D20. Peptides corresponding to the N-terminal 42, N-terminal 20 amino acids, and middle 20 amino acid sections could also form stable structures. The C-terminal segment did not protect any protons in D20. Interestingly, self assembly of the N-terminal 42 and C-terminal 16 amino acid peptides into a structure very close to that of the 60 amino acid domain was observed. The circular dichroism results reveals a large negative cotton effect at 198 nm that is characteristic of the proline-rich beta-turn helixes which consist predominantly of trans-proline. The intrinsic viscosity results suggest a non-random coil structure that is rod shaped. Our conclusion is that PRN60 forms a beta-turn helix and that this region of FeLV-gp70 is a separate folding domain of the retroviral surface unit glycoprotein. The unique conformational properties of PRN60 and its critical role as the predominant target for neutralizing antibody responses suggest that this peptide is a reasonable candidate for producing a synthetic peptide vaccine for FeLV.

Amino Acid Sequence↗
PubMed1994

Unusual basal cell carcinomas.

The typical features of basal cell carcinoma are well recognized. However, manifestations of uncommon and unusual basal cell carcinomas are less well known. In this review, unusual basal cell carcinomas are described based on their clinical characteristics, histologic appearance, behavior, and treatment.

Age Factors↗
PubMed1994

Liver regeneration after partial hepatectomy in the rat. Sequential events monitored by 31P-nuclear magnetic resonance spectroscopy and biochemical studies.

BACKGROUND: Little data exist wherein both the 31P nuclear magnetic resonance (NMR) signals and biochemical changes associated with hepatic regeneration after a 70% hepatic resection have been assessed simultaneously. EXPERIMENTAL DESIGN: Two groups of rats were used: one group underwent a 70% partial hepatectomy and the second underwent a sham operation. Both groups were followed sequentially for 192 hours by in vivo serial 31P-NMR spectroscopy of the liver and its phospholipid extracts. Liver injury and function were assessed by biochemical means. RESULTS: After surgery, a significant reduction in ATP and an increase in the phosphomonoester signal for the hepatectomized animals were noted as compared with the controls (p < 0.05). The phosphodiester content of the liver in the hepatectomized rats declined to nonmeasurable amounts in vivo. The nadir of ATP occurred 72 hours after surgery. The area of the phosphomonoester relative to an external reference of methylenediphosphonic acid peak increased steadily over the first 96 hours, whereas that of the area ratio of the inorganic phosphate/methylenediphosphonic increased over the first 72 hours posthepatectomy. The intracellular pH declined sharply in the first 3 days, followed by a gradual recovery over the next 5 days. Little change in the intracellular pH was observed for the control animals. A significant increase in the area of the phosphorylethanolamine relative to an internal reference of methylenediphosphonic and a reduction in the glycerophosphorylethanolamine and glycerophosphorylcholine peaks were noted during the first four post-hepatectomy days as measured by 31P-NMR of perchloric acid liver extracts (p < 0.05). CONCLUSIONS: It has been found that a concerted reduction in the intracellular ATP and intracellular pH coupled with an increase in inorganic phosphate and high levels of phosphorylethanolamine occur as a result of hepatic regeneration and the physiologic changes induced. These data demonstrated that a coordinated pattern of biochemical changes occur with and after hepatic regeneration. Moreover, NMR spectroscopy demonstrates an increase in phosphomonoesters and a decline in phosphodiesters during hepatic regeneration. These measures and, more specifically, the ratio of these two lipid classes may provide a biochemical snapshot of the regeneration status of the liver.

Adenosine Triphosphate↗
PubMed1994

The effects of functional electrical stimulation on shoulder subluxation, arm function recovery, and shoulder pain in hemiplegic stroke patients.

The purpose of this study was to evaluate the effectiveness of a functional electrical stimulation (FES) treatment program designed to prevent glenohumeral joint stretching and subsequent subluxation and shoulder pain in stroke patients. Twenty-six recent hemiplegic stroke patients with shoulder muscle flaccidity were randomly assigned to either a control group (n = 13; 5 female, and 8 male) or experimental group (n = 13; 6 female, and 7 male). Both groups received conventional physical therapy. The experimental group received additional FES therapy where two flaccid/paralyzed shoulder muscles (supraspinatus and posterior deltoid) were induced to contract repetitively up to 6 hours a day for 6 weeks. Duration of both the FES session and muscle contraction/relaxation ratio were progressively increased as performance improved. The experimental group showed significant improvements in arm function, electromyographic activity of the posterior deltoid, range of motion, and reduction in subluxation (as indicated by x-ray) compared with the control group. We concluded that the FES program was effective in reducing the severity of shoulder subluxation and pain, and possibly facilitating recovery of arm function.

Aged↗
PubMed1993

Biophysical characterization of one-, two-, and three-tandem repeats of human mucin (muc-1) protein core.

Until recently mucin tandem repeat protein cores were believed to exist in random-coil conformations and to attain structure solely by the addition of carbohydrates to serine and threonine residues. Matsushima et al. (Proteins Struct. Funct. Genet., 7: 125-155, 1990) recently proposed a model of the secondary structure of proline rich tandem repeat proteins that has challenged this idea, especially for the case of the human polymorphic epithelial mucin encoded by the muc-1 gene. We report here results of structural analyses of the muc-1 protein core by using synthetic peptide analogues. Synthetic peptides were prepared to correspond to one-, two-, and three-tandem repeats of muc-1. Results of one- and two-dimensional 1H NMR correlation spectroscopy on these peptides confirm that the muc-1 protein core is not a random-coil secondary structure. Long-lived amide protons are protected in D2O, and increasing spectral complexity in the region of the beta-protons of Asp2 and His 15 reveals that structural changes are occurring as the number of repeats increases. The greatest changes occur when the number of repeats increases from one to two. These results are supported by the reactivity of a panel of monoclonal antibodies raised against tumor associated muc-1 with these synthetic peptides in enzyme-linked immunosorbent assay. The primary immunodominant mucin epitope, PDTRP, does not appear to attain a native conformation in the single repeat peptide (20 amino acids, starting with P), but is expressed on peptides with multiple repeats. Intrinsic viscosity measurements of the peptide containing three repeats indicate that an ordered structure present in solution is rod shaped. The circular dichroism spectrum of the same peptide is dominated by proline in the trans conformation. These results are all consistent with the prediction that the muc-1 tandem repeat polypeptide core forms a polyproline beta-turn helix.

Amino Acid Sequence↗
PubMed1993

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated with glucose metabolism in human erythrocytes.

We have used [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has 13C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-13C]-, [2-13C]-, and [3-13C]D-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.

Adult↗
PubMed1993

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy.

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13 +/- 0.02 mumol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21 +/- 0.02 mumol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59 +/- 0.10 mumol/h per ml erythrocytes, compared to normal erythrocytes, 1.64 +/- 0.10 mumol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16 +/- 0.16 mumol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to stimulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.

Anemia, Sickle Cell↗
PubMed1993

Production of unmodified human adult hemoglobin in Escherichia coli.

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.

Amino Acid Sequence↗
PubMed1993

Inhibition of protein kinase C by alcohols and anaesthetics.

Despite almost a century of research, the mechanism of anaesthesia remains obscure and there is still no agreement on the location of the site(s) of action. Because the potencies of general anaesthetics increase in proportion to their solubility in olive oil, this led to a consensus that the site is within the cell membrane. This led to theories that lipid bilayer perturbation was the primary event, which was then transmitted to a membrane protein. But at the concentrations used clinically, such perturbations are small. A plausible site would be in or on ion channels at the synapse, where a number of modulatory effects have been described. A possible location for such a site would be at the protein-lipid interface. We report here that anaesthetics inhibit protein kinase C, a key component in signal transduction. The potency is a linear function of the octanol-water partition coefficient (the Meyer-Overton rule of anaesthesia). The effect was obtained in a lipid-free assay, implicating a hydrophobic site in the protein, supporting the contention that a (membrane) protein may be a target for anaesthetic interactions. In a lipid-dependent assay, a potential role of lipids in the protein-site model was demonstrated. The inhibition was absent in the isolated catalytic domain, suggesting that the site of inhibition is on the regulatory subunit, which is unique to protein kinase C.

Alcohols↗
PubMed1993

Contribution of hydrogen bonding to lipid-lipid interactions in membranes and the role of lipid order: effects of cholesterol, increased phospholipid unsaturation, and ethanol.

It is proposed that increased phospholipid unsaturation in membranes and perturbation by agents such as ethanol weaken interlipid hydrogen bonding involving water and that the process is independent of effects on lipid order. To investigate this, the rates of phospholipid desorption, as a measure of the strength of interlipid interactions, from "donor" lipid vesicles was determined. This was accomplished using (7-nitrobenzo-2-oxa-1,3-diazole-4-yl)aminohexanoate (C6-NBD) labeled phospholipids, the rate of desorption being followed from changes in fluorescence with time. The rates of desorption of the NBD-phospholipids from phosphatidylcholine (PC) donor vesicles was in the order phosphatidylcholine (PC) > phosphatidylserine (PS) > phosphatidylethanolamine (PE), the slower rates in the PS and PE reflecting direct interlipid hydrogen bonding. For PC, the interlipid hydrogen bonding was restricted to the "hydration layer", the network of hydrogen-bonded water molecules extending between phospholipid head groups. The rate of C6-NBD-PC desorption was elevated with higher levels of donor PC sn-2 unsaturation, due the increased head group spacing weakening the lipid-lipid interactions that occur via the hydration layer. Ethanol also increased the rate of NBD-phospholipid desorption from donor PC vesicles in the order PC > PS > PE, showing that PC interactions, here limited to the weaker hydrogen-bonded water molecule network, were more susceptible compared to stronger, direct interlipid hydrogen bonds involving PE and PS. The relative magnitude of the ethanol-induced increase in the desorption rate was amplified with higher levels of donor lipid sn-2 unsaturation. Cholesterol had little effect on the rate of phospholipid desorption.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗