Search PubMed⌕ Search

Biomedical subjects

C Ho

Publications and source records attributed to C Ho.

At least 145 records · Page 8Linked to original sources
PubMed1993

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates.

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (P(i)) to ATP ratio in normal livers as measured by 31P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the P(i)/ATP ratio of 18%, 40%, and 40%, respectively. 19F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to decreases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.

Adenosine Triphosphate↗
PubMed1993

Molecular cloning and sequence of a novel ommochrome-binding protein cDNA from an insect, Manduca sexta.

An ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta has recently been purified and characterized. A cDNA clone was isolated from a fifth instar larval cDNA expression library utilizing antiserum against OBP. Northern blot analysis of total fat body RNA detected a transcript of approximately 1.2 kilobases in fifth instar wandering larvae RNA. The complete nucleotide sequence of the 905-base pair cDNA insert was determined by the dideoxy chain termination method. The OBP cDNA encodes a polypeptide of 274 residues with a predicted molecular weight of 30,580 and with one consensus N-linked glycosylation site. Comparison of the NH2-terminal sequence of the mature protein and the cDNA sequence revealed a typical signal peptide of 18 amino acids. In wandering stage larvae, the OBP transcript appeared to be at least 250-fold less abundant than ribosomal RNA.

Amino Acid Sequence↗
PubMed1993

Intracellular labeling of T-cells with superparamagnetic contrast agents.

Isolated rat T-cells have been labeled intracellularly, using endocytosis uptake of two superparamagnetic contrast agents, AquaMag100 and BMS180549, which are both iron-oxide particles coated with dextran. No deterioration of cell proliferation response to mitogen stimulation was observed after labeling with either superparamagnetic contrast agent. AquaMag100 particles show aggregation and co-precipitation in culture media for T-cells. BMS180549 particles not only produce no observable aggregation or co-precipitation, but also have a higher efficiency for labeling T-cells than AquaMag100. The efficiency of cell labeling was determined by measuring the decrease in the spin-spin relaxation time of the water proton in cell samples containing 1 x 10(7) labeled T-cells/milliliter of 2% w/w gelatin. After optimization of the labeling procedures, a shortening of the spin-spin relaxation time by a factor of approximately 7 to 10 has been demonstrated. Under the present experimental conditions, the up-regulation of low density lipoprotein receptor does not increase the labeling efficiency by endocytosis. Our results suggest that intracellular labeling of specific cell types can be achieved with good efficiency and the labeled cells can be detected by magnetic resonance imaging in rat testicles in vivo.

Animals↗
PubMed1993

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.

DNA Mutational Analysis↗
PubMed1993

A rapid method for cloning mutagenic DNA repair genes: isolation of umu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a.

Genetic and physiological experiments have demonstrated that the products of the umu-like operon are directly required for mutagenic DNA repair in enterobacteria. To date, five such operons have been cloned and studied at the molecular level. Given the apparent wide occurrence of these mutagenic DNA repair genes in enterobacteria, it seems likely that related genes will be identified in other bacterial species and perhaps even in higher organisms. We are interested in identifying such genes. However, standard methods based on either DNA or protein cross-hybridization are laborious and, given the overall homology between previously identified members of this family (41 to 83% at the protein level), would probably have limited success. To facilitate the rapid identification of more diverse umu-like genes, we have constructed two Escherichia coli strains that allow us to identify umu-like genes after phenotypic complementation assays. With these two strains, we have cloned novel umu-like genes from three R plasmids, the IncJ plasmid R391 and two IncL/M plasmids, R446b and R471a.

Cloning, Molecular↗
PubMed1993

Effect of ethanol on energy status and intracellular calcium of Sertoli cells: a study using immobilized perfused cells.

The effects of ethanol on ATP, O2 consumption, and cytosolic ionized Ca2+ (Ca2+i) were studied in Sertoli cells isolated from the testes of 18- to 21-day-old rats. The cells were immobilized in agarose gel threads and perfused with Dulbecco's Modified Eagle's Medium. Intracellular ATP was determined by 31P nuclear magnetic resonance spectroscopy and enzymatic assay, Cai2+ was measured with the photoprotein aequorin, cell viability was assessed by trypan blue exclusion, and O2 consumption was monitored with a Clark electrode. Ethanol was used with or without pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole (MP). Perfusing the cells for 90 min with 500 mM ethanol produced a 50% reduction in the 31P nuclear magnetic resonance beta ATP signal, and pretreatment with 15 mM MP enhanced this decline of the beta ATP peak to 75%. Enzymatic measurements of ATP revealed that exposure to 500 mM ethanol reduced the ATP levels from 52 +/- 5 to 38 +/- 3 nmol/10(6) cells with MP pretreatment and to 28 +/- 4 nmol/10(6) cells without MP pretreatment (n = 5). Basal O2 consumption was 5.2 +/- 0.5 nmol/min.10(6) cells (n = 5), and it was reduced by ethanol or ethanol plus MP to 4 +/- 0.4 and 3.1 +/- 0.2, respectively (n = 5). The basal concentration of Cai2+ in Sertoli cells was 98 +/- 0.7 nM (n = 32). During perfusion with 500 mM ethanol, Cai2+ increased to 208 +/- 98 nM (n = 5) and was not modified further by the presence of MP. Perfusing the cells for 90 min with 500 mM ethanol with or without MP caused a decrease in cell viability from 93 +/- 2 to 76 +/- 3 and 67 +/- 3, respectively (n = 8). Exposure to 5 mM acetaldehyde produced only a minimal reduction in ATP, with no observable effect at lower concentrations, suggesting that the significant reductions in ATP, O2 consumption, and cell viability evoked by ethanol were not caused by acetaldehyde. These data suggest that ethanol is toxic to Sertoli cells, and its toxicity is not a result of ethanol metabolism.

Adenosine Triphosphate↗
PubMed1993

Epstein-Barr virus-derived vectors for transient and stable expression of recombinant proteins.

The transient expression level of foreign proteins in primate cells can be enhanced by incorporating the replication elements derived from Epstein-Barr virus (EBV). Specifically, we have constructed expression plasmids with the replication origin region (OriP) from EBV and an adenovirus-transformed cell line that expresses the EBV nuclear antigens-1 (EBNA-1). As EBV vectors can replicate as episomes in the nuclei, such vectors can have a stable transfection efficiency as high as 25% and provide a straightforward way of obtaining large amounts of recombinant proteins transiently or stably.

Antigens, Viral↗
PubMed1992

31P-NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity.

A system consisting of isolated rat hepatocytes immobilized in agarose threads continuously perifused with oxygenated Krebs-Henseleit (KH) solution has been found to maintain cell viability with excellent metabolic activity for more than 6 h. The hepatocytes were monitored by phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy at 4.7 Tesla, by measurement of oxygen consumption and by the leakage of lactate dehydrogenase (LD) and alanine aminotransferase (ALT). The data obtained were comparable to those found for an isolated perfused whole liver in vitro. The effects of allyl alcohol (AA), ethanol, and 4-acetaminophenol (AP) were examined. A solution of 225 microM AA perifused for 90 min caused the disappearance of the beta-phosphate resonance of adenosine triphosphate (ATP) in the 31P-NMR spectra, a 7-fold increase in LD leakage and a 70% reduction in oxygen consumption. Ethanol (1.0 M) perifused for 90 min reduced the beta-ATP signal intensity ratio by 20%, the phosphomonoester (PME) signal by 50% and inorganic phosphate (Pi) by 33% (P less than 0.05). AP (10 mM) caused only mild liver-cell damage. The results demonstrate that perifused immobilized hepatocytes can be used as a liver model to assess the effects of a wide range of chemicals and other xenobiotics by NMR spectroscopy.

Animals↗
PubMed1992

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores.

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.

Animals↗
PubMed1992

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.

Amino Acid Sequence↗
PubMed1992

Gramicidin conformational studies with mixed-chain unsaturated phospholipid bilayer systems.

The transition of gramicidin from a nonchannel to a channel form was investigated using mixed-chain phosphatidylcholine lipid bilayers. Gramicidin and phospholipids were codispersed, after removal of the solvents chloroform/methanol or trifluoroethanol which resulted in nonchannel and channel conformations, respectively, as confirmed using circular dichroism (CD). The fluorescence emission maxima of the nonchannel form were shifted toward shorter wavelengths by heating at 60 degrees C (for 0-12 h), which converted it to a channel form, again as confirmed by CD. The channel form did not respond to heat treatment. Heat treatment also increased the fluorescence anisotropy of the nonchannel gramicidin tryptophans. The rate of transition from the nonchannel to channel conformation was found to be faster if phosphatidylethanolamine was present in combination with phosphatidylcholine compared to phosphatidylcholine alone. Also, gramicidin in bilayers of the polyunsaturated 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine converted more rapidly compared to 1-palmitoyl-2-oleoylphosphatidylcholine. Using the fluorescence anisotropy of the membrane lipid probe 1,6-diphenyl-1,3,5-hexatriene, it was also shown that the motional properties of the surrounding lipid acyl chains differed for the channel and nonchannel gramicidin conformations. The possibility that lipids tending to favor the hexagonal phase (HII) would enhance the rate of the nonchannel to channel transition was supported by 31P NMR which revealed the presence of some HII lipids in the channel preparations. The results of this study suggest that gramicidin may serve as a useful model for similar conformational transitions in other more complex membrane proteins.

Circular Dichroism↗
PubMed1992

Multidimensional 1H and 15N NMR investigation of glutamine-binding protein of Escherichia coli.

Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp220 residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of L-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H epsilon 2 of His156 refines the earlier model where this particular proton forms an intermolecular hydrogen bond to the delta-carbonyl of L-glutamine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8-9 amino acid residues at a time. This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25,000.

Amino Acid Sequence↗
PubMed1992

In vivo studies of ethanol on prolactin and luteinizing hormone in rats and mice.

The interaction of ethanol (EtOH), prolactin (Prl) and luteinizing hormone (LH) was examined in two studies. In the first study, adult male C57B1/6J mice were given a single intraperitoneal injection of either vehicle or Prl at 5, 10 and 20 mg/kg and a significant dose-related suppression of ethanol consumption was found. This injection did not cause any differences in food intake or body weight. Additionally, a 5 mg/kg dose of Prl was also given to adult male Long Evans Hooded rats and, similarly, there was a significant suppression of ethanol consumption. In a second study, when rats were given a free choice between water and 5% EtOH, three subgroups were found regarding the amount of EtOH consumption: low, medium and high. After 2 weeks of free choice, hypothalamic, but not serum Prl and LH levels, were significantly increased in EtOH-imbibing groups compared to controls. These findings suggest important interactions between EtOH consumption and ambient levels of Prl and LH.

Alcohol Drinking↗
PubMed1992

Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data. Altered hydration at the protein-lipid interface could affect conformation, thereby offering a new route by which membrane protein functioning may be modified.

Apoproteins↗
PubMed1992

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.

Amino Acid Sequence↗
PubMed1992

Development of extended-release solid dispersions of nonsteroidal antiinflammatory drugs with aqueous polymeric dispersions: optimization of drug release via a curve-fitting technique.

Extended-release solid dispersions of nonsteroidal antiinflammatory drugs were prepared by using aqueous polymeric dispersions of Eudragit RS30D and Eudragit RL30D as the inert carriers. The effects of different polymer ratios of Eudragit RS30D and Eudragit RL30D, different particle sizes, and different combination of various formulations of solid dispersions on the in vitro release kinetics of drugs from the dosage forms were investigated. A computer curve-fitting process was developed to choose the optimum formulation of the solid dispersion with the desired drug release profile. This process might offer the advantages of efficiency and simplicity in the formulation development of extended-release solid dispersions.

Acrylic Resins↗
PubMed1992

Magnetic resonance imaging in assessing cartilage changes in experimental osteoarthrosis of the knee.

Cartilage degeneration in osteoarthrosis (OA) of the knee generally is believed to precede osseous abnormalities. Because cartilage abnormalities are not readily detected by routine radiography, we investigated the role of magnetic resonance imaging (MRI) in assessing cartilage damage in a goat model for OA. Four goats had the anterior cruciate ligament of one knee severed surgically to create instability and accelerate OA. Two goats each were killed at 4 and 6 weeks, respectively, after walking on the unstable knees. MRI of the knees was performed with Hybrid fat suppression sequences. The images were correlated with gross anatomic sections and histologic analysis of the knees. On gross examination, the unstable knees showed rapid development of thinning, surface irregularity, and focal defects of the cartilage. These findings correlated well with abnormalities detected on the MRIs. In addition, areas of decreased signal intensity in cartilage correlated with histologic evidence of degenerative changes in the cartilage substance, including fragmentation, fibrillary and eosinophilic changes, and chondrocyte proliferation, indicating attempted cartilage repair. Precise correlation of pathologic and MRI data, however, was lacking, related in part to inability to match perfectly the level and orientation of the gross section with that on the MRI examination.

Animals↗