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Biomedical subjects

C Yu

Publications and source records attributed to C Yu.

At least 163 records · Page 9Linked to original sources
PubMed2000

[Effect of alginate purity on microencapsulated hepatocyte overgrowth in peritoneal transplantation].

OBJECTION: To study the effect of alginate purity on microcapsule overgrowth in peritoneal transplantation. METHODS: Alginate was purified by filtration and chloroform/butanol extraction. Using purified and crude alginate to make two kinds of microencapsulated hepatocytes (MHs), we transplanted 5 ml of each kind of MHs into the peritoneal cavities of rats. After 2 weeks, 1, 2, and 6 months, MHs were washed out to study overgrowth of MH, histology, enzyme and albumin of hepatocytes. Human lymphocytes transformation was measured by 3H-TdR incorporation with two kinds of alginate solutions. RESULTS: The amount of retrieved MHs was larger in the purified than in the crude ( P < 0.01). In the purified groups, most hepatocytes had normal features of morphology, enzymatic histochemistry, and albumin immunohistochemistry staining after one month of transplantation, but were damaged in the crude groups only 2 weeks later. Microcapsules were smooth, regular and had no overgrowth during 6 months in the purified but overgrowth one month later after transplantation in the crude. The liquid scintillation counting (cpm) was lower in the purified than in the crude. CONCLUSION: The purified alginate could reduce the overgrowth of MH and improve the function of hepatocytes significantly.

Alginates↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro.

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. ACAT-1 may function as an allosteric enzyme. We took a multifaceted approach to investigate the subunit composition of ACAT-1. When ACAT-1 with two different tags were co-expressed in the same Chinese hamster ovary cells, antibody specific to one tag caused co-immunoprecipitation of both types of ACAT-1 proteins. Radioimmunoprecipitations of cells expressing the untagged ACAT-1 or the 6-histidine-tagged ACAT-1 yielded a single radiolabeled band of predicted size on SDS-polyacrylamide gel electrophoresis. These results show that ACAT-1 exists as homo-oligomers in intact Chinese hamster ovary cells. We solubilized HisACAT-1 with the detergent deoxycholate or CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), performed gel filtration chromatography and sucrose density gradient centrifugations in H(2)O and D(2)O, and determined the Stokes radii and sedimentation coefficients of the HisACAT1-detergent complexes. The estimated molecular mass of HisACAT-1 is 263 kDa, which is 4 times that of the HisACAT-1 monomer (69 kDa). Finally, cross-linking experiments in intact cells and in vitro show that the increase in cross-linker concentrations causes an increase in size of the HisACAT-1-positive signals, forming material(s) 4 times the size of the monomer, supporting the conclusion that ACAT-1 is a homotetrameric enzyme.

Animals↗
PubMed1999

Differential expression of carbohydrate blood-group antigens on rat taste-bud cells: relation to the functional marker alpha-gustducin.

An afferent nerve fiber supplying a taste bud receives input from several taste receptor cells, yet is predominantly responsive to one of the classic taste qualities (salt, acid, sweet, or bitter). This specificity requires recognition between taste receptor cells and nerve fibers that may be mediated by surface markers correlating with function. In an effort to identify potential markers, we used immunofluorescence and confocal microscopy to examine expression of the oligosaccharide blood-group antigens Lewis(b), A, and H type 2 in taste buds of the rat oral cavity. We compared the distributions of these antigens with that of alpha-gustducin, a G-protein subunit implicated in responses to sweet- and bitter-tasting substances. The A and Lewis(b) antigens were present only on spindle-shaped cells whose apical processes reached the taste pore. These antigens were not present on epithelial cells surrounding taste buds, and Lewis(b) was not found elsewhere in the digestive tract. Lewis(b) and A were not removed by lipid extraction, suggesting that they are present on glycoproteins rather than glycolipids. All Lewis(b)-positive cells expressed alpha-gustducin, but only a fraction of alpha-gustducin-positive cells expressed Lewis(b). The fraction of taste-bud cells expressing Lewis(b) decreased in the order: vallate papillae > foliate papillae > nasoincisor duct. The epiglottis had almost no taste-bud cells that expressed Lewis(b). The A antigen appeared on taste-bud cells that also expressed alpha-gustducin in the order: foliate and vallate papillae > nasoincisor duct and epiglottis > fungiform papillae. In addition, the A antigen was present on many cells that lacked alpha-gustducin in foliate and vallate papillae. In vallate papillae, cells expressed either A or Lewis(b), but not both. Lewis(b) appears to be restricted to differentiated light cells that also express alpha-gustducin and may be involved in intercellular interactions of these cells.

Animals↗
PubMed1999

Diminished aqueous microviscosity of tumors in murine models measured with in vivo radiofrequency electron paramagnetic resonance.

Using very low frequency in vivo electron paramagnetic resonance (EPR), we have compared, for the first time, the average microviscosity of the total aqueous compartment of murine fibrosarcomas and that of normal leg tissue in a living animal. EPR spectra from dissolved nitroxide spin probes report the solvent microviscosity. The tumor aqueous microviscosity, 1.8 +/- 0.1 centipoise, was significantly lower than that of the corresponding normal tissue, 2.9 +/- 0.3 centipoise, a difference of 38 +/- 7%. These results confirm the commonly observed increase in the water proton transverse relaxation times (T2) in magnetic resonance imaging of hyperproliferative states, for example, malignancy. The specificity of the localization of the EPR signal indicates a substantial portion of the T2 increase seen in magnetic resonance imaging derives from decreased bulk-water viscosity. The effect of this microviscosity differences may be the basis of several physiological differences between tumors and normal tissues which could confer a growth rate advantage to tumor tissue.

Animals↗
PubMed1999

Dosimetric comparison of three photon radiosurgery techniques for an elongated ellipsoid target.

PURPOSE: To examine the dosimetric differences among three radiosurgery techniques: gamma knife, linac multiple arcs, and conformally-shaped static fields. METHODS AND MATERIALS: A simulated target was taken to be a prolate ellipsoid, 25 mm in diameter, 35 mm in length, centrally located in a three-dimensional (3D) model of a patient head taken from MR images. Single isocenter linac treatment plans were developed, 9 portals for the static shaped field technique, and a 7-arc plan for the multiple arc method. A total of 13 isocenters with 3 different collimators were used in the gamma knife plan. RESULTS: At dose levels from 25% to 50% of the reference dose, multiple arc and shaped-field plans treated a greater volume than the gamma knife plan. The linac plans, however, delivered the dose more homogeneously across the target volume as compared to the gamma knife plan. For the dose levels between 50-100%, the shaped fields and gamma knife plan have a similar dose distribution, and treated slightly less volume than the multiple arc plan. CONCLUSION: For a target of limited volume and essentially any shape, one can obtain closely conformal dosimetry with the gamma knife. For a regular-shaped target, the single isocenter multiple arc technique gives a more homogenous dose distribution within the target. Static shaped fields offer an alternative radiosurgery technique, with dosimetry similar to the multiple arc method, applicable to targets of any shape.

Head↗
PubMed1999

Stable mixed hematopoietic chimerism in dogs given donor antigen, CTLA4Ig, and 100 cGy total body irradiation before and pharmacologic immunosuppression after marrow transplant.

Stable mixed chimerism can be established in dogs given a sublethal dose of 200 cGy total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) for 28 and 35 days, respectively, after dog leukocyte antigen-identical marrow transplantation. Most likely, the role of pretransplant TBI was to provide host immunosuppression, since stable mixed chimerism was also achieved in MMF/CSP-treated dogs when 450 cGy irradiation, targeted to cervical, thoracic, and upper abdominal lymph nodes, was substituted for TBI. When TBI was reduced from 200 to 100 cGy, all grafts were rejected within 3 to 12 weeks. Here, we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with help of the fusion peptide CTLA4Ig, which blocks T-cell costimulation through the B7-CD28 signal pathway. Accordingly, recipient T cells were activated with intravenous (IV) injections of 10(6) donor peripheral blood mononuclear cells (PBMC)/kg per day on days -7 to -1 before 100 cGy TBI, with concurrent administration of CTLA4Ig 4 mg/kg/d IV. All 7 dogs so treated showed initial mixed chimerism. Two rejected their allografts after 8 and 20 weeks, respectively, and survived with autologous marrow recovery; 1 mixed chimera was unevaluable because of death at 3 weeks from intussusception; and 4 showed persisting mixed chimerism, including unirradiated marrow and lymph node spaces, for now more than 46 to 70 weeks after transplant. Data support the hypothesis that stable marrow allografts can be established by combining nonmyeloablative pretransplant host immunosuppression with posttransplant host and donor cell immunosuppression using MMF/CSP.

Abatacept↗
PubMed1999

Mutations of human topoisomerase II alpha affecting multidrug resistance and sensitivity.

Two mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.

Adenosine Triphosphate↗
PubMed1999

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.

Amides↗
PubMed1999

Stable mixed hematopoietic chimerism in dog leukocyte antigen-identical littermate dogs given lymph node irradiation before and pharmacologic immunosuppression after marrow transplantation.

Stable mixed donor/host hematopoietic chimerism can be accomplished in dog leukocyte antigen (DLA)-identical littermate dogs given sublethal (200 cGy) total-body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) after transplant (Blood 89:3048, 1997). Studies were based on the hypothesis that drugs that prevent graft-versus-host disease (GVHD) after transplant also suppress host-versus-graft (HVG) reactions and thereby enhance engraftment. Here, we asked whether pretransplant TBI provided marrow space for the graft to home or caused host immunosuppression. To address the questions, recipients were given pretransplant irradiation to cervical, thoracic, and abdominal lymph nodes (except pelvis), DLA-identical littermate marrow grafts, and MMF/CSP posttransplant. Six dogs that received 450 cGy irradiation showed initial engraftment. Two rejected their grafts after 8 and 18 weeks, 1 died with GVHD and engraftment, and 3 are alive as mixed chimeras after 57 to 97 weeks. Four dogs given 200 cGy irradiation also showed initial engraftment, but rejected their grafts after 10 to 18 weeks. Mixed chimerism was present in nonirradiated marrow and lymph node spaces and involved granulocytes, T cells, and monocytes. While other explanations are possible, results seem consistent with the hypothesis that pretransplant radiation provides host immunosuppression, and grafts can create their own marrow space. These data set the stage for the development of novel transplant regimens that substitute immunosuppressive for cytotoxic agents.

Animals↗
PubMed1999

Cloning and mapping of the XRN2 gene to human chromosome 20p11.1-p11.2.

The Dhm1 gene is the mouse homologue of the dhp1(+) gene of Schizosaccharomyces pombe, which is involved in homologous recombination and RNA metabolism, such as RNA synthesis and RNA trafficking, in S. pombe. Complementation analysis showed the Dhm1 gene on a multicopy plasmid can rescue the temperature-sensitivity mutation of dhp1(ts) and the lethality of the dhp1 null mutation. This finding suggests that Dhm1 has a function in mouse similar to that of dhp1(+). The human homologue of this gene, XRN2, has been identified. A 3.6-kb transcript of XRN2 was detected in 16 tissues examined and was more abundant in testis. By radiation hybrid panel mapping, the XRN2 gene was localized to chromosome 20p11.1-p11.2 between markers D20S180 and D20S871.

Adult↗
PubMed1999

GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression.

The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals.

Animals↗
PubMed1999

Secondary structure formation is the earliest structural event in the refolding of an all beta-sheet protein.

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding.

Cobra Neurotoxin Proteins↗
PubMed1999

Cellular expression of alpha-gustducin and the A blood group antigen in rat fungiform taste buds cross-reinnervated by the IXth nerve.

Although taste buds are trophically dependent on their innervation, cross-reinnervation experiments have shown that their gustatory sensitivities are determined by the local epithelium. Both the gustatory G-protein, alpha-gustducin, and the cell-surface carbohydrate, the A blood group antigen, are expressed by significantly fewer fungiform than vallate taste cells in the rat. In these experiments, one side of the anterior portion of the tongue was cross-reinnervated by the IXth nerve in order to determine whether the molecular expression of taste bud cells is determined by the epithelium from which they arise or by the nerve on which they are trophically dependent. The proximal portion of the IXth nerve was anastomosed to the distal portion of the chorda tympani (CT) nerve using fibrin glue (IX-CT rats). Control animals had the CT cut and reanastomosed using the same technique (CT-CT rats), or had the CT avulsed from the bulla and resected to prevent regeneration (CTX rats). The animals survived for 12 weeks postoperatively, and the tongues were removed, stained with methylene blue, and the fungiform taste pores counted on both sides. Tissue from the anterior 5 mm of the tongue was cut into 50-microm sections, which were incubated with antibodies against alpha-gustducin and the human blood group A antigen. In both CT-CT and IX-CT rats, there was regeneration of fungiform taste buds, although in both groups there were significantly fewer taste buds on the operated side of the tongue. The normal vallate papilla had a mean of 8.37 alpha-gustducin-expressing cells and 5.22 A-expressing cells per taste bud, whereas the fungiform papillae contained 3.06 and 0.23 cells per taste bud, respectively. In both CT-CT and IX-CT rats there was a normal number of cells expressing alpha-gustducin or the A antigen in regenerated taste buds; in the CTX animals there was a significant decrease in the expression of these markers. These results demonstrate that the molecular phenotype of taste bud cells is determined by the local epithelium from which they arise and not by properties of the innervating nerve.

ABO Blood-Group System↗
PubMed1999

Binding of nucleotide triphosphates to cardiotoxin analogue II from the Taiwan cobra venom (Naja naja atra). Elucidation of the structural interactions in the dATP-cardiotoxin analogue ii complex.

Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na+,K+-ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP.CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the N- and C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na+,K+-ATPase is also proposed.

Circular Dichroism↗
PubMed1999

Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress.

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H2O2 were shown to produce high molecular weight (HMW) DNA fragments approximately 50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.

DNA Damage↗
PubMed1999

Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus.

Evidence indicates that the carboxy-terminal cytoplasmic domain of glucose transporter 4 (GLUT4) is important for the regulation of GLUT4 in muscle and adipocytes. We cloned from a human skeletal muscle cDNA library a 34-kDa protein which interacts with GLUT4 C-terminal cytoplasmic domain in a two-hybrid system and also with GLUT4 C-terminus synthetic peptide in an in vitro binding assay. This protein, called YP10, showed a high degree (>90%) of sequence homology with l-3-hydroxyacyl-CoA dehydrogenase (HAD) and had a dehydrogenase activity similar to pig heart HAD, which was inhibited by GLUT4 C-terminus synthetic peptide. An antiserum raised against pig heart HAD also reacted with YP10. Western blot analysis using this antiserum revealed abundant immunoreactivity only in the mitochondria- and plasma membrane-enriched fractions of rat adipocytes. Northern blots revealed that YP10 mRNA is most abundant in skeletal and heart muscle. These findings suggest that YP10, a HAD isoform, interacts with GLUT4 at the plasma membrane and may play a role in cross-talk between glucose transport and fatty acid metabolism.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Structurally homologous toxins isolated from the Taiwan cobra (Naja naja atra) differ significantly in their structural stability.

Cardiotoxin and neurotoxin analogues isolated from snake venom sources are highly homologous proteins (>50% homology) with similar three-dimensional structures but exhibit drastically different biological properties. In the present study, we compare the conformational stability of cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX), a neurotoxin analogue, from the Taiwan cobra (Naja naja atra), using circular dichroism spectroscopy and hydrogen-deuterium (H/D) exchange techniques in conjunction with two-dimensional NMR methods. Contrary to expectations, it is found that CTX III and CBTX differ significantly in their structural stabilities. The three-dimensional structure of CBTX is less stable than that of CTX III. The amide protons of residues at the N- and C-terminal ends of the CTX III molecule are strongly protected against H/D exchange, implying that the terminal ends of the molecule are bridged together by significant numbers of hydrogen bonds. However, in CBTX, amide protons at the terminal ends of the molecule do not exhibit an significant protection against H/D exchange. Comparison of the protection factors of the various amide protons in CTX III and CBTX reveals that the extraordinary stability of CTX III stems from the strong network of interactions among the residues at the N- and C-terminal ends and also due to the tight and ordered packing of the nonpolar residues involved in the triple-stranded, anti-parallel, beta-sheet segment of the molecule.

Animals↗