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C Chen

Publications and source records attributed to C Chen.

At least 775 records · Page 43Linked to original sources
PubMed1996

The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor.

We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor.

Animals↗
PubMed1996

Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala8,37]-echistatin gamma with three disulfide bonds by 2D-NMR and computer graphics.

An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin gamma, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin gamma, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin gamma, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin gamma contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin alpha1 which differs from echistatin gamma by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin alpha 1 even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin gamma, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.

Amino Acid Sequence↗
PubMed1996

G(o)-2 protein mediates the reduction in Ca2+ currents by somatostatin in cultured ovine somatotrophs.

1. Somatotroph-enriched cells (up to 85%) were obtained from ovine pituitary glands by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the reduction in Ca2+ currents by 10 nM somatostatin (SRIF). 2. The whole-cell configuration of the patch-clamp technique was employed to study the membrane Ca2+ currents with K+ ions replaced by Cs+ and the addition of K+ and Na+ channel blockers in bath and pipette solutions. 3. A significant reduction in Ca2+ currents was obtained in response to local application of SRIF (10 nM) but vehicle application had no effect. 4. Intracellular dialysis of antibodies to alpha(o), alpha(i)-1-2, or alpha(i)-3 subunits of G proteins into the cells via patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis did not modify resting voltage-gated Ca2+ currents across the cell membrane. 5. Dialysis of anti-alpha(o) antibodies significantly attenuated the reduction in Ca2+ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti-alpha(i)-1-2 nor anti-alpha(i)-3 antibodies diminished the effect of SRIF on Ca2+ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the alpha(o) subunit mRNA (alpha(o) ASm, for alpha(o) common) or against the alpha(i)-3 subunit mRNA (alpha(i)-3 AS) blocked expression of alpha(o) or alpha(i)-3 subunits in the cells, respectively, as assessed by fluorescent staining with anti-alpha(o) or anti-alpha(i)-3 antibodies 48 h after dialysis. 7. Dialysis of alpha(o) ASm, but not alpha(i)-3 AS, significantly diminished the inhibitory effect of SRIF on Ca2+ currents. This effect of alpha(o) ASm dialysis occurred within 12 h after dialysis and reached a maximum at 48 h; partial recovery was seen at 72 h. 8. Antisense oligonucleotides specific for alpha(o)-1 (alpha(o)-1 AS) or alpha(o)-2 (alpha(o)-2 AS) were dialysed into somatotrophs and only alpha(o)-2 AS significantly attenuated the inhibition of Ca2+ currents by SRIF. 9. We conclude that the G(o)-2 protein mediates the effect of SRIF on Ca2+ currents in ovine somatotrophs in primary culture.

Animals↗
PubMed1996

Renal artery perfusion modifies ischemia/reperfusion injury.

Renal ischemic and reperfusion injury is a significant complication of major aortic and renovascular surgery. The delivery of a preservative agent just prior to reperfusion of an ischemic kidney may decrease the reperfusion injury. The purpose of this study was to evaluate the effects of renal artery perfusates delivered at the termination of an ischemic period. Five groups of rats were evaluated. All rats underwent left nephrectomy. The right kidney was made ischemic for 45 min by occlusion of the renal artery and vein. Ischemic control animals had no renal artery perfusion. Nonischemic control animals had no renal vessel occlusion or perfusion. The other three groups were perfused during the final 4 min of ischemia with one of the following: normal saline (NS), phosphate-buffered saline (PBS), or anti-ICAM-1-antibody (mAb). The blood urea nitrogen (BUN), serum creatinine (Cr), and renal histopathologic injury of each group were compared. The ischemic control group had significantly better renal function than the group perfused with NS or mAb at 72 hr. There was no significant difference between the ischemic control and PBS groups in renal function or morphologic injury. It is concluded that none of the perfusates in the study had protected the kidney from ischemic and reperfusion injury. NS delivered in this manner was injurious to the kidney.

Animals↗
PubMed1996

Local infusion of FGF-saporin reduces intimal hyperplasia.

The recent conjugation of the potent ribosome-inactivating protein saporin (SAP) with basic fibroblast growth factor (FGF2) to form recombinant (r)FGF2-SAP permits increased selectivity of this mitoxin for cells exhibiting upregulated FGF receptors. Systemic administration of rFGF-SAP in therapeutic doses, however, may be associated with significant liver toxicity. In this blinded study, we used a local boundary layer infusion approach to increase local drug concentration while minimizing the risk of side effects. Six dogs underwent bilateral carotid endarterectomies. Expanded polytetrafluoroethylene infusion devices, blindly primed with rFGF2-SAP to one artery or vehicle to the contralateral vessel, were anastomosed proximal to the injured segments so that each animal served as its own control. rFGF2-SAP (2 microgram/kg/day) or vehicle (5 microl/hr) was continuously delivered for 14 days from an osmotic reservoir, through the wall of the graft infusion device. Euthanasia was carried out at 14 days and the processed arteries were blindly analyzed for intimal thickening and cellular proliferation. All dogs survived until sacrifice with no clinical side effects. Liver function tests at euthanasia were not significantly altered when compared to baseline values. Intimal area in rFGF2-SAP-treated vessels averaged 0.31 +/- 0.10 mm2 versus 0.57 +/- 0.24 mm2 in the control segments (P = 0.02), a relative reduction of 46%. Cell proliferation, however, was not significantly different at 14 days postendarterectomy (2.40 +/- 1.31% vs 2.39 +/- 0.45%). From this study it can be concluded that locally delivered rFGF2-SAP reduces intimal hyperplasia and that the boundary layer infusion strategy is an effective means for delivering high local drug concentration while minimizing systemic drug effects.

Amino Acid Sequence↗
PubMed1996

Tenascin: a potential role in human arteriovenous PTFE graft failure.

To investigate the involvement of tenascin (TN) in human neointimal hyperplastic lesion formation, we studied 12 human arteriovenous (AV) polytetrafluoroethylene (PTFE) loop grafts removed at the time of graft revision. Immunoperoxidase technique was used to determine TN and proliferating cell nuclear antigen (PCNA) as an index of cell proliferation. The venous anastomotic neointimal hyperplastic lesion was analyzed as three layers, each one-third of the thickness. TN was distributed in the neointima as follows: (1) in the luminal layer, all lesions were intensely stained; (2) in the middle layer, 9 of 12 (75%) lesions had moderate reactivity; (3) in the deep layer near the PTFE grafts, 3 of 12 (25%) lesions were strongly stained and 5 of 12 (42%) had moderate reactivity; and (4) 90 +/- 6% of microvessels within the neointima showed intense periendothelial staining. PCNA indices were as follows: luminal layer, 29 +/- 11%; middle layer, 9 +/- 5%; deep layer near the graft, 9 +/- 7%; and microvessel containing intimal fields, 67 +/- 4%. The cell proliferation rate was significantly higher in the luminal layer than in the middle or deep layers of neointima (P < 0.05). The PCNA index in microvessel-containing intimal fields was three to eight times that of avascular fields (P < 0.001). These data demonstrate that TN is distributed in a pattern similar to that of cell proliferation in human neointimal hyperplastic lesions. The results suggest that TN expression may have a potential role in neovascularization and neointimal growth in human AV PTFE graft failure.

Adult↗
PubMed1996

Irreversible binding of cis-(+)-3-methylfentanyl isothiocyanate to the delta opioid receptor and determination of its binding domain.

Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized. SUPERFIT inhibited [3H]diprenorphine binding with much higher affinity for the delta than the mu or kappa receptor. Pretreatment with SUPERFIT followed by extensive washing reduced delta binding with an IC50 value of 7.1 nM, yet it did not affect mu and kappa binding up to 0.1 microns. The reduction in delta binding by SUPERFIT pretreatment was due to a decrease in Bmax with no change in Kd. These results indicate that SUPERFIT is a highly selective delta irreversible ligand. We then determined the region in the delta receptor that confered binding selectivity for SUPERFIT by examining its binding to six mu/delta chimeric receptors. SUPERFIT bound to delta, mu/delta 1 (amino acids mu 1-94/delta 76-372), delta/mu 3 (delta 1-134/mu 154-398), and delta/mu 4 (delta 1-187/mu 207-398) receptors with high affinity but to mu, delta/mu 1 (delta 1-75/mu 95-398), mu/delta 3 (mu 1-153/delta 135-372), and mu/delta 4 (mu 1-206/delta 188-372) receptors with low affinity. Pretreatment with SUPERFIT potently inhibited [3H]diprenorphine binding to delta, mu/delta 1, delta/mu 3, and delta/mu 4 but affected binding to mu, delta/mu 1, mu/delta 3, and mu/delta 4 only at much higher concentrations. Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the delta receptor is important for selective binding of SUPERFIT.

Amino Acid Sequence↗
PubMed1996

Determination of three-dimensional solution structure of waglerin I, a toxin from Trimeresurus wagleri, using 2D-NMR and molecular dynamics simulation.

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 omega torsion angel restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

Amino Acid Sequence↗
PubMed1996

Mass spectrometric analyses of beta-ketolactone oligomers, macrocyclic or catenane structures?

Mass spectrometry is used to develop an analytical method for a new class of organic oligomeric material, beta-ketolactones. Three different series with varying oligomeric sizes are examined. The oligomers may form at least two different structures, a macrocyclic and a catenane ring. Fast atom bombardment coupled with Fourier transform mass spectrometry provides a rapid and convenient method that provides both molecular weight information and abundant fragment ions that are structurally relevant. All the compounds are found to work well with the mass spectrometric analysis. Electrospray ionization coupled with triple-quadrupole mass spectrometry was also evaluated and found to yield results that are similar to FAB. On the basis of the FAB and the low-energy collisionally activated dissociation spectra, we conclude that these compounds are macrocyclic rings and not catenanes.

Chromatography, Liquid↗
PubMed1996

Mechanism of impaired glucose-potentiated insulin secretion in diabetic 90% pancreatectomy rats. Study using glucagonlike peptide-1 (7-37).

Chronic hyperglycemia causes a near-total disappearance of glucose-induced insulin secretion. To determine if glucose potentiation of nonglucose secretagogues is impaired, insulin responses to 10(-9) M glucagonlike peptide-1 (GLP-1) (7-37) were measured at 2.8, 8.3, and 16.7 mM glucose with the in vitro perfused pancreas in rats 4-6 wk after 90% pancreatectomy (Px) and sham-operated controls. In the controls, insulin output to GLP-1 was > 100-fold greater at 16.7 mM glucose versus 2.8 mM glucose. In contrast, the increase was less than threefold in Px, reaching an insulin response at 16.7 mM glucose that was 10 +/- 2% of the controls, well below the predicted 35-40% fractional beta-cell mass in these rats. Px and control rats then underwent a 40-h fast followed by pancreas perfusion using a protocol of 20 min at 16.7 mM glucose followed by 15 min at 16.7 mM glucose/10(-9) M GLP-1. In control rats, fasting suppressed insulin release to high glucose (by 90%) and to GLP-1 (by 60%) without changing the pancreatic insulin content. In contrast, in Px the insulin response to GLP-1 tripled in association with a threefold increase of the insulin content, both now being twice normal when stratified for the fractional beta-cell mass. The mechanism of the increased pancreas insulin content was investigated by assessing islet glucose metabolism and proinsulin biosynthesis. In controls with fasting, both fell 30-50%. In Px, the degree of suppression with fasting was similar, but the attained levels both exceeded those of the controls because of higher baseline (nonfasted) values. In summary, chronic hyperglycemia is associated with a fasting-induced paradoxical increase in glucose-potentiated insulin secretion. In Px rats, the mechanism is an increase in the beta-cell insulin stores, which suggests a causative role for a lowered beta-cell insulin content in the impaired glucose-potentiation of insulin secretion.

Animals↗
PubMed1996

Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections.

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin-paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a 'test battery' approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120 degrees C, 100 degrees C, and 90 degrees C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100 degrees C proved most effective. Both fresh and routinely processed formalin-paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin-paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin-paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.

Cryopreservation↗
PubMed1996

Pharmacokinetics of retinoids in women after meal consumption or vitamin A supplementation.

These studies were conducted to evaluate the pharmacokinetics of several retinoids after meal consumption or vitamin A supplementation to establish a reference for future assessment of teratogenic risks of retinoid therapeutic agents. In the first study, 36 healthy young female volunteers consumed single meals containing vitamin A amounts ranging from 1,305 to 169,474 IU. In the second study, 24 other female volunteers took vitamin A supplements at a dose level of 5,000, 10,000, or 25,000 IU/day for 60 days. Plasma concentrations of tretinoin, isotretinoin, 4-oxo-tretinoin, and 4-oxo-isotretinoin in samples collected during the studies were analyzed using a high-performance liquid chromatography method with ultraviolet detection. Pharmacokinetic parameters for the retinoids were calculated using model-independent methods. Plasma concentrations of tretinoin were not altered by meal consumption or vitamin A supplementation. Plasma levels of 4-oxo-tretinoin were below the assay detection limit (0.3 ng/mL) in the majority of samples collected throughout the studies. Linear relationships between dose and maximum concentration (Cmax) and dose and area under the concentration-time curve (AUC) for isotretinoin and 4-oxo-isotretinoin were derived from data from the meal study. For the most bioavailable formulation used in the supplement study, daily ingestion of 5,000 IU of vitamin A caused increases of 141 +/- 53% and 171 +/- 77% from baseline in the 24-hour AUCs of isotretinoin and 4-oxo-isotretinoin, respectively. Dose-related increases in systemic exposure to retinoids were observed after ingestion of vitamin A by means of a meal or a supplement. Findings from these studies can be used as a basis for future safety evaluations of retinoid compounds.

Adult↗
PubMed1996

Estimation of in vivo proton intrinsic and cross-relaxation rate in human brain.

By using on-resonance binomial pulse saturation of the immobile component in tissues and by monitoring the time development of the longitudinal decay of the free water magnetization, it has been shown that it is possible to estimate physical model parameters not directly measurable by conventional methods. Since pulsed saturation is easy and safe to implement in a clinical MRI machine, it should be possible to estimate these parameters in vivo by the same means. In this article, we report the results of such estimates of the parameters of the principal components (gray and white matter) in brains of two normal volunteers.

Adult↗
PubMed1996

The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana.

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, 3JNH alpha and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel beta-sheets and three alpha-helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 alpha-protons located in the loop-1 region, an alpha 2 helix, and three beta-strands, (beta 1, beta 3 and beta 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.

Amino Acid Sequence↗
PubMed1996

Normothermic renal artery perfusion: a comparison of perfusates.

Hypothermia and preservative perfusates have been used to decrease ischemic renal injury. This study was performed to identify the preservative function of perfusates independent of the effects of hypothermia. Rats underwent 45 minutes of renal ischemia. Rectal and renal parenchyma temperatures were monitored and maintained within 1 degree C of normal. Perfusates were University of Wisconsin solution (UW), Euro-Collins solution, normal saline solution, and Ringer's lactate solution. A nonperfused ischemic control and a nonischemic control group were also evaluated. Parameters evaluated included serum creatinine and blood urea nitrogen levels, renal ischemic injury grade, renal weight, and gross appearance of the injured kidney. Rats treated with UW solution were found to have a significantly lower creatinine, blood urea nitrogen, and injury grade than the other three perfused groups. The external gross appearance of the UW-treated kidneys was normal, whereas that of the other groups demonstrated moderate to severe injury. Although the mean right/left renal weight difference of the UW-treated group was lower than that of the other three groups, this was not statistically significant. Under normothermic conditions in rats, UW solution affords significant renal protection from ischemia. Euro-Collins, normal saline, and Ringer's lactate solutions display no significant protective effect.

Adenosine↗