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Biomedical subjects

Y F Chen

Publications and source records attributed to Y F Chen.

At least 73 records · Page 4Linked to original sources
PubMed2001

[High expression of rabbit sperm membrane protein rSP10 in Escherichia coli and preparation of its specific antisera].

To study the position and immunogenicity of rSP10, the rabbit rsp10 gene which did not include sequences coding for signal peptides was inserted into expression vector pET30a. An in-frame fusion protein was made such that a His6 stretch was produced at the N terminus of re-rSP10. High expression was obtained, the amount of re-rSP10 up to 67% in the total bacterial protein. The re-rSP10 was purified by DEAE chromatography and the yield of purified re-rSP10 was approximately 50 micrograms/mL of culture. Western blotting analysis of re-rSP10 with rabbit polyclonal sera raised against rabbit sperm membrane protein showed that the synthesized antigen possessed immunogenicity of rSP10. Specific antisera against re-rSP10 was induced using purified re-rSP10 as an antigen. The motility of capacitated sperms were affected but no aggregation was observed.

Animals↗
PubMed2001

[Exogenous pGH gene localization on chromosomes of the transgenic pigs].

The pUC19-OMT plasmids were cut by Ssp 1 and the products in 900 bp were recovered from low-melting agarose gel and used as probes which were labeled by Digoxigenin. After being denatured, the probes were dropped on the chromosome samples which were also denatured to anneal with them. The anti-digoxigenin antibodies labeled with colloidal gold were used to act with the chromosome samples. In order to localize the exogenous pGH genes(porcine growth hormone gene) on chromosomes detected with optical microscope and improve the sensitivity, digoxigen gold signals are amplified by silver precipitation. After calculating the number of silver grains on every chromosome under the optical microscope, we analyzed the data with statistical methods. The results show that the integrating sites of exogenous pGH genes are very different among the positives. However, it is clear that the exogenous genes in one are always of the tendency to integrate in one specific site on a certain chromosome. These data are of great significance for studying the site-specific integration and the expression efficiency of exogenous genes in the future research.

Animals↗
PubMed2001

Epithelial growth within the lamellar interface after laser in situ keratomileusis.

We report on a case of 36-year-old man with excessive myopia and amblyopia who underwent laser in situ keratomileusis (LASIK). The surgery was performed smoothly, and visual acuity improved greatly. The patient was lost to follow-up in our clinic until 2 months later when he returned because of blurred vision. Epithelial growth within the lamellar interface was noted which disclosed white, plaquelike, confluent opacities within the lamellar interface on the visual axis. Topography showed an irregular astigmatism overlying the entrance to the pupil. The flap was lifted at the flap edge, but it was difficult to lift it around the area of epithelium growth within the lamellar interface under an operating microscope. The epithelial tightly adhered to the lamellar interface, so surgery was aborted because of the possibility of flap perforation. Prednisolone acetate 1% was used for one month, but the epithelial growth within the lamellar interface persisted. LASIK has the impressive potential to correct refractive errors. Epithelial growth within the interface after LASIK, although usually a benign complication, can lead to a poor outcome and even permanent vision loss.

Adult↗
PubMed2001

Endothelial cell loss induced by phacoemulsification occurs through apoptosis.

BACKGROUND: Cornea endothelial cells are nondividing cells containing pumping function which is crucial for cornea clarity and integrity. Endothelial cell loss occurs after cataract surgical procedures such as phacoemulsification. The authors hypothesize that endothelium damage occurs through apoptosis. METHODS: Ultrasound was achieved by placing a phacoemulsification probe in the anterior chamber and delivering 0% or 50% of maximum power for 2.5 min. The corneal tissue was harvested immediately, and at 1 and 7 days after the operation. Corneal tissue was stained by hemotoxylin and eosin (H&E) and evaluated by light microscopy. Endothelium apoptosis was monitored using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay to detect DNA fragmentation. RESULTS: In corneas which underwent phacoemulsification with 0% energy, no cell loss or apoptosis was identified immediately, 1 or 7 days after the operation. Likewise, in corneas exposed to 50% energy, no cell loss or apoptosis was detected immediately after phacoemulsification. However, minimal amount of cell loss but prominent apoptosis was detected with the TUNEL assay 1 day after the operation, whereas significant cell loss but no apoptosis was detected 7 days after the operation by H&E stain. CONCLUSIONS: These results demonstrate that corneal endothelial cell loss induced by ultrasound damage occurs through apoptosis.

Animals↗
PubMed2000

Estrogen attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of osteopontin production in vascular smooth muscle cells.

BACKGROUND: Previous in vitro studies have suggested that estrogen attenuates the vascular injury response by modulating vascular smooth muscle cell (VSMC) expression of soluble factor(s) directing migration of adventitial fibroblasts. Previous in vivo studies have established a role for osteopontin (OPN) and its integrin receptors after vascular injury. In this study, we examined OPN expression in activated VSMCs, its modulation by estrogen, and its effects on adventitial fibroblast migration. In addition, the relative functional roles of beta(1)- and beta(3)-integrin-matrix interactions were examined. METHODS AND RESULTS: Primary cultures of VSMCs and adventitial fibroblasts were derived from female Sprague-Dawley rats. Serum-activated VSMCs expressed high levels of OPN mRNA and secreted protein that was effectively inhibited by estrogen treatment (10(-7) mol/L). Compared with VSMCs, fibroblasts expressed similar levels of integrins alphanu and beta(1) and higher levels of integrin-beta(3). Exogenous OPN (5.0 to 40 microg/mL) directed fibroblast migration in a dose-dependent fashion. Anti-beta(3)-integrin antibody (F11) pretreatment markedly inhibited adventitial fibroblast migration directed by exogenous OPN or VSMC-conditioned medium in a dose-dependent manner. In contrast, anti-beta(1)-integrin antibody (Ha2/5) did not affect fibroblast migration. Similarly, pretreatment with either linear or cyclic RGD peptides (10 to 1000 micromol/L) inhibited fibroblast migration directed by OPN or VSMC-conditioned medium in a dose-dependent manner. CONCLUSIONS: These observations suggest that estrogen indirectly attenuates integrin-beta(3)-dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.

Animals↗
PubMed2000

A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains.

Two series of peptides that specifically bind to the extracellular domain of the alpha chain of the human interleukin-5 receptor (IL-5Ralpha), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor alpha/beta heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor alpha chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Ralpha extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R alpha chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.

Amino Acid Sequence↗
PubMed2000

Determination of unbound cephalothin in rat blood by on-line microdialysis and microbore liquid chromatography.

A method of analysis for the determination of unbound cephalothin in rat blood has been developed. The method was fully automated using an on-line microdialysis procedure. A microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats to examine the unbound cephalothin level in the rat blood after cephalothin administration (50 mg/kg, i.v.). Dialysates were directly input to a liquid chromatographic system using an on-line injector. Samples were eluted with a mobile phase containing methanol-acetonitrile-100 mM monosodium phosphate (pH 5.0) (20:20:60, v/v). The UV wavelength was set at 254 nm for monitoring the analyte. Using the retrograde method, at infusion concentrations of 1 and 5 microg/ml of cephalothin, the in vivo microdialysis recoveries were 48.4+/-4.5% and 52.9+/-4.7% for the rat blood (n=6). Intra- and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.01 through 10 microg/ml. Pharmacokinetic parameters were calculated from the recovery corrected dialysate concentrations of cephalothin versus time data. The results suggest that the pharmacokinetics of unbound cephalothin in blood fitted best to the two-compartmental model following cephalothin administration (50 mg/kg, i.v.).

Animals↗
PubMed2000

Estrogen-induced vasoprotection is estrogen receptor dependent: evidence from the balloon-injured rat carotid artery model.

BACKGROUND: Previous studies have shown that estrogen (E2) is vasoprotective in multiple animal models of vascular injury, including mice with homologous disruptions of either the alpha or beta isoforms of the estrogen receptor (ER) gene, calling into question the ER dependency of the vasoprotective effect. This study used ICI 182,780, a nonselective ER antagonist, to test the hypothesis that the vasoprotective effect of E2 in the rat carotid injury model is ER mediated. METHODS AND RESULTS: Intact female Sprague-Dawley rats were divided into 4 groups and treated with the nonselective ER antagonist ICI 182,780 (ICI; 0.5, 1.5, or 5 mg. kg(-1). d(-1), subcutaneously [S.C.]) or vehicle, beginning before balloon injury of the right common carotid artery and continuing for 14 days afterward. Four groups of ovariectomized rats (OVX) were treated with 17beta estradiol (E2) (20 microgram. kg(-1). d(-1), S.C.) alone or combined with ICI 5 mg. kg(-1). d(-1), S.C.; with ICI 5 mg. kg(-1). d(-1) alone; or with vehicle according to a similar protocol. Two weeks after injury, rats were killed, and the carotid arteries were evaluated for neointima formation using morphometric analysis. ICI 182,780 blunted the E2-related protective effect and increased neointima formation in injured carotid arteries of intact female rats in a dose-dependent fashion. ICI had no effect on neointima formation in OVX, but addition of ICI to E2 in OVX blocked the inhibitory effect of exogenous E2 on neointima formation. CONCLUSIONS: These results indicate that the vasoprotective effect of E2 in the balloon-injured rat carotid artery model is mediated by ER.

Animals↗
PubMed2000

Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries.

BACKGROUND: Clinical and experimental evidence suggest that the adventitia participates in the response to endoluminal vascular injury. The current study used a direct approach to test the hypothesis that, after balloon injury of the rat carotid artery, adventitial fibroblasts migrate in a luminal direction and contribute to neointima formation. METHODS AND RESULTS: Primary syngeneic adventitial fibroblasts were stably transduced with retroviral particles coordinating expression of beta-galactosidase (LacZ) and introduced into the adventitia of right carotid arteries of rats immediately after balloon injury. At defined times after injury and fibroblast implantation, rats were euthanized, and arterial tissue was examined for detection of LacZ mRNA (reverse transcription polymerase chain reaction), DNA (polymerase chain reaction), and in situ enzymatic activity. LacZ expression was detected in the media 5 days postinjury and in both media and neointima at 7, 10, and 14 days postinjury. LacZ was undetectable in injured vessels that had not been seeded with transduced fibroblasts and was restricted to the adventitia in seeded vessels that were not injured. CONCLUSIONS: These observations provide direct demonstration of adventitial fibroblast migration into neointima of arteries after endoluminal injury.

Animals↗
PubMed2000

Measurement and pharmacokinetics of unbound 20(S)-camptothecin in rat blood and brain by microdialysis coupled to microbore liquid chromatography with fluorescence detection.

To characterize the pharmacokinetics of protein-free camptothecin in blood and brain we implanted microdialysis probes into the jugular vein and striatum of rats for unbound drug sampling and determination. Camptothecin (2 or 5 mg/kg, i.v., n=6) was then administered from the femoral vein, and microdialysates were collected from blood and brain of both sites and assayed by a validated microbore scale high-performance liquid chromatographic method. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5) with a flow-rate 0.05 ml/min. The fluorescence response for camptothecin was observed at excitation and emission wavelengths of 360 and 440 nm, respectively. Pharmacokinetic parameters were calculated from the corrected data for dialysate concentrations of camptothecin versus time. The results suggest that the pharmacokinetics of unbound camptothecin in blood and brain can be fitted best to a two- and one-compartment model, respectively. Camptothecin rapidly entered the extracellular fluid of brain striatum at 10 min following camptothecin administration.

Animals↗
PubMed2000

Determination of chlorogenic acid in rat blood by microdialysis coupled with microbore liquid chromatography and its application to pharmacokinetic studies.

To investigate the pharmacokinetics of unbound chlorogenic acid, a sensitive microbore liquid chromatographic method for the determination of chlorogenic acid in rat blood by microdialysis has been developed. A microdialysis probe was inserted into the jugular vein of male Sprague-Dawley rats, to which chlorogenic acid (20, 40, 60 or 80 mg/kg, i.v.) had been administered. On-line microdialysate was directly injected into a microbore column using a methanol-100 mM sodium dihydrogenphosphate (30:70, v/v, pH 2.5 adjusted with orthophosphoric acid) as the mobile phase and ultraviolet detection at 325 nm. The method is rapid, easily reproduced, selective and sensitive. The limit of detection for chlorogenic acid was 0.01 microg/ml and the limit of quantification was 0.05 microg/ml. The in vivo recovery of the chlorogenic acid of the microdialysis probe, based on a 5 microg/ml standard, was approximately 49-65% (n=6). The disposition of chlorogenic acid at each dose was best fitted to a two-compartment pharmacokinetic model. The area under the concentration curve increased greater than in direct proportion with the dose and terminal disposition become much slower as the dose was increased. The results indicated that the pharmacokinetics of unbound chlorogenic acid in rat blood is non-linear.

Animals↗
PubMed2000

Concurrent quantification and pharmacokinetic analysis of cefotaxime in rat blood and brain by microdialysis and microbore liquid chromatography.

A simple but effective coupling of microdialysis and microbore liquid chromatograph with UV detection technique was applied to the simultaneous studying of the pharmacokinetics of cefotaxime in both the peripheral compartment and central nervous system. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5) pumped through a C18 microbore column at a flow-rate of 0.05 ml/min. Detection of cefotaxime was set at a UV wavelength of 254 nm. Microdialysis probes were inserted into the jugular vein and striatum of the rat. Following stabilization of microdialysate levels, rats received cefotaxime (20 mg/kg, i.v., n=6) via the femoral vein, and complete concentration versus time profiles for blood and striatum were constructed. The results indicated that cefotaxime rapidly (within 10 min) entered the extracellular fluid of brain striatum following intravenous administration. Noncompartmental pharmacokinetics analysis indicated that the area under the concentration versus time ratio of cefotaxime in rat brain and blood was 6.9%, suggesting appreciable blood-brain barrier penetration. The method was relatively simple, imposed minimal physiological perturbance as it involved no body fluid consumption and sampled in particular protein-unbound drugs, generally believed to be the active fraction.

Animals↗
PubMed2000

Simultaneous determination of cefazolin in rat blood and brain by microdialysis and microbore liquid chromatography.

A sensitive microbore liquid chromatographic method combined with the minimally invasive technique of microdialysis was devised for simultaneously and continuously monitoring the levels of unbound blood and brain cefazolin in rats. Microdialysis probes were inserted into the jugular vein and brain striatum for blood and brain sampling, respectively. Chromatographic conditions consisted of a mobile phase of methanol-acetonitrile-100 mM monosodium phosphoric acid (20:10:70, v/v, pH 4.5) pumped through a microbore reversed-phase column at a flow rate of 0.05 mL/min. The ultraviolet detection wavelength was set at 270 nm. An on-line design allowed direct and continuous analysis of protein-free samples in the dialysate. Microdialysis probes, being home-made, were screened for acceptable in vivo recovery. Chromatographic resolution and detection were validated for response linearity as well as intra-day and inter-day variabilities. This method was then applied to pharmacokinetic profiling of protein unbound cefazolin in both the blood and brain following intravenous administration (10 mg/kg, i.v., n = 6). Rapid appearance of cefazolin in the rat brain striatal dialysate following drug injection suggested good blood-brain barrier penetration. According to a non-compartmental pharmacokinetics model, the area under the concentration (AUC) vs time ratio of cefazolin in rat brain and blood was 6%.

Animals↗
PubMed2000

Attenuation of pulmonary vascular hypertension and cardiac hypertrophy with sitaxsentan sodium, an orally active ET(A) receptor antagonist.

Effects of sitaxsentan (TBC11251), an orally active, highly selective antagonist of endothelin A receptors, were examined on the development and maintenance of pulmonary hypertension, pulmonary vascular remodeling, and cardiac hypertrophy in the rat. The pulmonary vasoconstrictor response to acute hypoxia (10% O(2)for 90 min) was prevented with sitaxsentan (5 mg/kg infused iv 10 min prior to the onset of hypoxia) while BQ-788 (a specific endothelin B receptor antagonist) was without effect. The same dose of sitaxsentan delivered iv 50 min after the onset of hypoxia reversed the established pulmonary vasoconstriction. In a 2-week model of hypoxia using 10% O(2), treatment with sitaxsentan (15 mg/kg per day in drinking water) attenuated pulmonary hypertension and the associated right ventricular hypertrophy, and prevented the remodeling of small pulmonary arteries (50-100 microM) without affecting systemic arterial blood pressure or heart rate. Institution of sitaxsentan treatment (15 and 30 mg/kg per day in drinking water) for 4 weeks after 2 weeks of untreated hypoxia produced a significant, dose dependent reversal of the established pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling despite continued hypoxic exposure. Sitaxsentan blocked increased plasma endothelin levels in the prevention protocol but did not affect the established elevated levels in the intervention study. Sitaxsentan dose dependently (10 and 50 mg/kg per day in the drinking water) attenuated right ventricular systolic pressure, right heart hypertrophy, and pulmonary vascular remodeling observed 3 weeks after a single subcutaneous injection of monocrotaline. These findings support the hypothesis that endothelin-1 plays a significant role in the development of pulmonary hypertension, pulmonary vascular remodeling, and the associated cardiac hypertrophy, and further suggest that specific endothelin-A receptor blockade may be useful in the treatment of pulmonary hypertension of diverse etiologies.

Animals↗
PubMed2000

Peptide growth factor phytosulfokine-alpha contributes to the pollen population effect.

Density-dependent pollen germination and tube growth in vitro is a well-documented phenomenon, termed the pollen population effect, but far less is known about its molecular basis. We present evidence to support phytosulfokine-alpha [Y(SO3H)IY(SO3H)TQ; PSK-alpha] as a native bioactive factor contributing to this effect. Mature pollen grains of Nicotiana tabacum L. var. macrophylla were incubated in liquid medium for 2 h. Pollen germination frequency increased in a density-dependent manner from 625 to 46,000 grains/ml. Conditioned medium, obtained from the medium of pollen cultured at a density of 10,000 pollen grains/ml for 12 h, promoted the germination of pollen cultured at a low density (625 grains/ml). A rabbit antiserum against PSK-alpha specifically inhibited the promotive effect of conditioned medium. Quantification by enzyme-linked immunosorbent assay showed that the conditioned medium contained 0.4 nM of PSK-alpha. Exogenous PSK-alpha also stimulated pollen germination in the low-density culture. These results indicate that PSK-alpha is an important regulator involved in the pollen population effect.

Cells, Cultured↗