Biomedical subjects

Y C Pan

Publications and source records attributed to Y C Pan.

At least 19 recordsLinked to original sources

PubMed2000

Increased axonal regeneration through a biodegradable amnionic tube nerve conduit: effect of local delivery and incorporation of nerve growth factor/hyaluronic acid media.

The authors emphasize the possible pharmacological enhancement of axonal regeneration using a specific growth factor/ extracellular media incorporated in a biodegradable nonneural nerve conduit material. They investigated the early effects on nerve regeneration of continuous local delivery of nerve growth factor (NGF) and the local incorporation of hyaluronic acid (HA) inside a newly manufactured nerve conduit material from fresh human amnionic membrane. Human amnionic membrane contains important biochemical factors that play a major neurotrophic role in the nerve regeneration process. The process of manufacturing a nerve conduit from fresh human amnionic membrane is described. This nerve conduit system was used in rabbits to bridge a 25-mm nerve gap over 3 months. NGF was released locally, over 28 days, at the distal end of the tube via a system of slow release, and HA was incorporated inside the lumen of the tube at the time of surgery. NGF/HA treatment promoted axonal regeneration across the amnionic tube nerve conduit (8,962 +/- 383 myelinated axons) 45% better than the nontreated amnionic tube group (6,180 +/- 353 myelinated axons). The authors demonstrate that NGF/HA media enhances additional axonal regeneration in the amnionic tube nerve conduit. This result is secondary to the effect of the amnion promoting biochemical factors, in combination with the NGF/HA effect on facilitating early events in the nerve regeneration process.

Amnion↗

PubMed2000

[Repair of flexor-tendon injury in children's finger using microsurgical technique].

OBJECTIVE: To improve the clinical result of repair on flexor tendon injury, and recover the defected finger function in children as far as possible. METHODS: From January 1990 to October 1997, 12 cases with flexor tendon injury were repaired by microsurgical technique, sutured by modified Kessler method with 3/0 or 5/0 nontraumatic thread and followed by invering suture of the gap edge with 7/0 or 8/0 nontraumatic thread after debridement. Appropriate functional practice was performed postoperatively. RESULTS: All the defected fingers were healed by first intention. Followed up 6 months to 1 year, there was excellent in 7 cases, better in 4 cases, moderate in 1 case and 91.67% in excellent rate according to the TAM standard of International Hand Committee. CONCLUSION: The important measures to improve the clinical result in children's flexor tendon injury are prompt and accurate diagnosis and repair of the injured tendon by microsurgical technique, and effective postoperative functional practice.

Child↗

PubMed1999

Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract.

A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions.

Cell Extracts↗

PubMed1997

Positional isomers of monopegylated interferon alpha-2a: isolation, characterization, and biological activity.

The success of recombinant interferon alpha in the clinic in part is limited by two properties of the protein: short serum half-life and immunogenicity. To improve these properties, interferon alpha-2a was conjugated with polyethylene glycol (PEG-5000). A homogeneous preparation of monopegylated interferon alpha-2a was subjected to vigorous analytical and activity characterization. A newly developed ampholyte-free chromatofocussing-like cation-exchange HPLC method utilizing a sulfopropyl resin was used to separate the monopegylated protein into 11 species. Peptide mapping, sequencing, and mass spectrometric analyses indicated that these species are positional isomers where each isomer represents a single polymer molecule conjugated to one specific lysine residue. No species with a modification at the amino terminus was observed. All 11 isomers show antiviral and antiproliferative activities in the same range as the parent monopegylated interferon alpha-2a.

Animals↗

PubMed1997

Functional assessment of tibial-nerve recovery in the cat using gait analysis: preliminary study.

The purpose of this study was to investigate gait-pattern changes after complete tibial nerve lesion in the cat, and to observe whether nerve repair could reverse some of the changes. In six cats, a 5-cm segment of the tibial nerve was transected. The nerve gap was then repaired with nerve autograft in three animals and was unrepaired in three as controls. The walking patterns of the cats were videotaped, and the hip, knee, ankle, and metatarsophalangeal joint angles were measured at the beginnings of the F, E1, E2, and E3 phases of the step cycle. Two weeks after surgery, abnormal gait patterns were observed, and four gait parameters (E3.Hip, E3.Ankle, E3.M-P, and F.Ankle) were found to be statistically significantly different from normal. Six months after surgery, the nerve-graft group had gait-parameter values approaching normal, while the control group showed no measurable improvement. Correspondingly, electrophysiologic testing revealed considerable nerve regeneration in the nerve-graft group but not in the control group. It was concluded that these gait parameters can be used as valid functional indices to evaluate the degree of tibial nerve recovery in the cat model.

Action Potentials↗

PubMed1997

Results of laser tissue soldering in vasovasostomy and epididymovasostomy: experience in the rat animal model.

UNLABELLED: The use of microsurgical techniques (vasovasostomy and epididymovasostomy) for vasectomy reversal has now enabled surgeons to perform both procedures with certainly acceptable success rates. However, these operations are technically demanding and require special training in microsurgery. PURPOSE: A new method of performing these procedures using laser tissue soldering is described and results are evaluated. Laser tissue soldering is different from laser welding in that it involves the laser activation of a protein solder with a dye specific for the specific wavelength of laser light; therefore, surrounding tissue is not affected by the laser. MATERIALS AND METHODS: Ten rats underwent bilateral vasovasostomy and eleven underwent bilateral epididymovasostomy. In each rat, a sutured anastomosis was performed on one side while laser tissue soldering was performed on the other. Animals were sacrificed after one month and anastomoses were evaluated for patency and presence of sperm granulomas. Histologic analysis was also performed. RESULTS: Patency rates were 8/10 (80%) for sutured vasovasostomy versus 9/10 (90%) for the laser technique. Epididymovasostomy patency rates were 8/11 (73%) for sutured versus 9/11 (82%) for the laser technique. Mean operative times were significantly shorter for lasered anastomoses when compared to controls. The frequency of granuloma formation did not significantly differ between laser and control groups. CONCLUSIONS: Laser tissue soldering resulted in similar patency when compared to a conventional 2 layered sutured anastomosis while decreasing operative time. In addition, since fewer sutures are placed, the laser method is less technically demanding.

Animals↗

PubMed1996

Ankle stance angle: a functional index for the evaluation of sciatic nerve recovery after complete transection.

This study attempted to develop a motor functional index, ankle stance angle (ASA), to assess rat sciatic nerve regeneration subsequent to autografting. ASA, 50 degrees in normal rats, is the ankle joint angle at the mid-stance phase of the gait cycle. In a nerve graft group, a 1-cm segment of the right sciatic nerve was transected and then repaired with nerve autograft. In an ungrafted group, the nerve gap was left unrepaired. ASA measured 4 months after surgery was statistically significantly larger in the nerve graft group (36 degrees) than in the ungrafted group (22 degrees). The results suggest that ASA is more sensitive than sciatic function index in detecting functional recovery after a complete sciatic nerve lesion. ASA also showed a significant correlation with the passive range of ankle joint motion and gastrocnemius muscle weight. The study concluded that ASA is a reliable index for assessment of regeneration of rat sciatic nerve after a complete lesion. The intra-rater reliability (r = 0.97 and 0.90) and inter-rater reliability (r = 0.85) tests performed support the conclusions.

Animals↗

PubMed1995

Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity.

IL-12, a heterodimeric cytokine, consists of two disulfide-linked subunits, p40 and p35. We investigated the role of p40 in ligand binding and signal transduction by expressing this subunit alone in COS cells. Culture media of the transfected COS cells exhibited specific dose-dependent binding to KIT225/K6 cells, a human T cell line that expresses IL-12R. Analysis of the culture media by SDS-PAGE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-linked homodimers. The two p40 species were purified and identified by N-terminal sequencing and proteolytic peptide mapping. Characterization of the p40 proteins for binding and bioactivity showed that both the p40 monomer and dimer inhibited 125I-labeled IL-12 binding to IL-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) of 20 to 70 ng/ml, was at least 20-fold more effective than the monomer. Although neither the monomer nor the dimer stimulated human PHA-blast proliferation, the 80-kDa dimer inhibited IL-12-induced proliferation in a dose-dependent manner with an IC50 of 65 ng/ml. The results suggest that the IL-12 p40 subunit contains the essential epitopes for receptor binding. However, a proper conformation required for high affinity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit. When p40 is associated with a p35 subunit, the heterodimer acts as an agonist mediating biologic activity. However, when p40 associates with another p40, the homodimer behaves as an antagonist in vitro.

Animals↗

PubMed1995

Immunoregulatory effects of indomethacin on rats with trauma.

In this article, we investigated changes of immune functions and immunoregulatory effects of indomethacin on rats with trauma. The results show that spontaneous suppressor T cell activity of spleen significantly increased and Interleukin I production and DNA synthesis capacity of splenocytes markedly decreased in rats with trauma. Indomethacin could markedly improve immune function, decreased spontaneous suppressor T cell activity and prompted Interleukin 2 production and DNA synthesis capacity of splenocytes.

Abdominal Injuries↗

PubMed1995

Experimental orthotopic transplantation of vascularized skeletal allografts: functional assessment and long-term survival.

Vascularized skeletal tissue allografts would greatly expand the domain of reconstructive surgery. Few studies to date have examined the functional aspects of these allografts or their long-term fate. An orthotopic transplant model of rat distal femur and surrounding muscular cuff was developed to assess graft function in fracture healing and weight bearing. Isografts (RT1l to RT1l, n = 40), weak-barrier allografts (RT1l to RT1lv, n = 40), and strong-barrier allografts (RT1l to RT1n, n = 40) were transplanted. As the histocompatibility barrier increased between the donor and recipient animals, the graft viability and performance deteriorated according to radiographic, histologic, and immunologic analyses. Administration of cyclosporine led to survival of strong-barrier allografts similar to that of isografts. A long-term study of these allografts (RT1l to RT1n) was then performed on various immunosuppressive regimens. After an initial 10-week course of cyclosporine to achieve bony union and remodeling, subsequent cessation (n = 20) or intermittent "pulsing" (n = 20) of the immunosuppressant was insufficient in maintaining graft survival. However, graft viability and function were preserved through 1 year on continuous daily cyclosporine (n = 32). There was no evidence of host renal or hepatic toxicity by serum chemistry or histologic sections. Thus long-term survival of functional skeletal allografts was achieved in this orthotopic model without significant host toxicity from immunosuppression.

Animals↗

PubMed1994

Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses.

Electrophoresis, Polyacrylamide Gel↗

PubMed1994

A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules.

Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.

Adrenal Medulla↗

PubMed1994

Purification and characterization of a high-molecular-weight form of recombinant human interleukin-2.

During purification of recombinant Interleukin-2 (rIL-2) by reversed-phase HPLC, early fractions are discarded due to the presence of an unidentified form of rIL-2. A procedure has been developed to isolate and purify this unidentified form of rIL-2. The purification process involves two chromatography steps and utilizes a Bakerbond Carboxy-Sulfon (CS) column under two different conditions. This material, designated as a high-molecular-weight form of rIL-2 (HMWrIL-2), exhibits lower mobility during SDS-PAGE and has a pI which is approximately one unit less than that of rIL-2, but has similar bioactivity to rIL-2. Structural analysis through enzymatic cleavage, HPLC peptide mapping, mass spectrometry, sequencing, and amino acid composition revealed that the difference between these two proteins is a C-terminal extension of 11 amino acids. This extension could be the result of a nonstandard translation event.

Amino Acid Sequence↗

PubMed1993

Electroblotting proteolytic products from native gel for direct N-terminal sequence analysis: an approach for studying protein-protein interaction.

Proteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis. This method, in conjunction with limited proteolysis and N-terminal sequencing, has been used to study the molecular interactions between native protein molecules. The interaction between recombinant interleukin-2 (rIL-2) and its receptor (rIL-2R alpha) was examined as a model system. The working strategy consists of (i) proteolysis of rIL-2R alpha and rIL-2R alpha/rIL-2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis. Results have indicated that the exon 3 encoded region in rIL-2R alpha is sensitive to proteolysis regardless whether it is complexed with rIL-2 or not. This suggests that no major conformational changes occur in rIL-2R alpha during interaction with rIL-2. This electroblotting approach is, therefore, useful for studying protein-protein interaction in solution.

Animals↗

PubMed1993

Limited tryptic digestion of recombinant human interleukin-2: structure-binding relationships with the alpha chain of the interleukin-2 receptor.

The surface topography and structural features of interleukin-2 (IL-2) in relation to its interaction with the alpha subunit of its receptor (IL-2R alpha) have been probed by limited tryptic digestion followed by detailed structural analyses. Four sensitive cleavage sites in IL-2 (Lys8, Lys9, Lys35, and Arg38) were identified as surface amino acids, suggesting that they are potential binding sites for IL-2R alpha. To examine the involvement of these residues in IL-2R alpha binding, a truncated IL-2 molecule lacking the amino-terminal residues through Arg38 was generated and it was found to be incapable of binding IL-2R alpha in a solid-phase receptor binding sequencing assay. These studies have led to the conclusion that the IL-2R alpha contact region of IL-2 includes residues Lys35 and Arg38. This finding is supported by the refined three-dimensional structure of IL-2 in which these residues are located outside of the compact bundle of four helices and thus are readily available for interaction with IL-2R alpha.

Amino Acid Sequence↗

PubMed1993

A simple technique for harvesting standardized skin grafts in the rodent.

A simple technique for harvesting skin grafts of predetermined size, shape, and thickness in rodents is described. In this technique, the donor skin is immobilized and tensed by means of a tongue depressor, or similar template, inserted into the loose areolar tissue below the panniculus, stretching the overlying skin to permit easy dermatome harvesting of a skin graft.

Animals↗

PubMed1993

[Analysis of keratin filament structural transformation in human epithelial cells].

Upon 2-3 hours cold treatment (0 degrees C), the keratin filaments of some PcaSE-1 cells and BEL-7404 cells are partly transformed into granular aggregates. But such structural transformation does not occur in HeLa cells and CNE cells. By rewarming (37 degrees C) cells within 15-30 minutes, this structural changes of keratin filaments in PcaSE-1 cells and BEL-7404 cells are readily reversed. In contrast, in HeLa cells and CNE cells, keratin filaments are transformed into granula aggregates during mitosis, but the keratin filament network in PcaSE-1 cells and BEL-7404 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. Results suggest that the above two types of keratin filament structural transformation might be induced by different factors. Our results also indicate that: (1) PcaSE-1 cells treated with colchicine alone or with combination of colchicine and cytochalasin D does not cause granular aggregates of keratin filaments. However, after depolymerization of microtubules with colchicine, the response of the cells to cold treatment is intensified. (2) The aggregate formation during cold treatment is unrelated to whether epithelial cells contain two different type intermediate filaments or not. (3) Epithelial cells preextracted with Triton X-100 do not induce granular aggregate formation of keratin filaments upon cold treatment. (4) The structural transformation upon cold treatment may be a characteristic of keratin filaments of certain epithelial cell lines.

Cold Temperature↗

PubMed1992

Endothelial monocyte-activating polypeptide II. A novel tumor-derived polypeptide that activates host-response mechanisms.

An important means by which tumor cells influence the vasculature is through the production of soluble mediators altering vascular properties. A approximately 22-kDa polypeptide was purified to homogeneity from conditioned medium of murine methylcholanthrene A (meth A) fibrosarcoma cells by ion-exchange chromatography and preparative sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), based on its ability to induce tissue factor procoagulant activity in endothelial cells (ECs). The final product migrated as a broad band on reduced and nonreduced SDS-PAGE and had an unique amino-terminal sequence. This meth A-derived polypeptide modulated EC coagulant properties through the induction of tissue factor, induced monocyte migration and tissue factor expression, and was also chemotactic for granulocytes. Injection of the polypeptide into mouse footpads resulted in an inflammatory response with tissue swelling and polymorphonuclear leukocyte infiltration. The ability of this mediator to activate ECs and monocytes has led us to name it EMAP II (endothelial monocyte-activating polypeptide). EMAP II is distinct from a previously described approximately 40-kDa meth A-derived polypeptide termed EMAP I. Through its potential to activate host effector mechanisms, EMAP II could contribute to the biology of immunogenic tumors, such as the meth A fibrosarcoma.

Amino Acid Sequence↗