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Biomedical subjects

P W Cheng

Publications and source records attributed to P W Cheng.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of epidermal growth factor, transferrin, and insulin on lipofection efficiency in human lung carcinoma cells.

Poor transfection efficiency is the major drawback of lipofection. We showed previously that addition of transferrin (TF) to Lipofectin enhanced the expression of a reporter gene in HeLa cells by 120-fold and achieved close to 100% transfection efficiency. The purpose of this study was to determine whether TF and other ligands could improve the efficiency of lipofection in lung carcinoma cells. Confluent A549, Calu3, and H292 cells were transfected for 18 hours with a plasmid DNA (pCMVlacZ) using Lipofectin plus TF, insulin, or epidermal growth factor as the vector. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (light units/microg protein) and the percentage of blue cells following 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside staining. Lipofectin supplemented with epidermal growth factor yielded the largest enhancement of lipofection efficiency (< or =23-fold over that by Lipofectin alone) in all three cell lines. Insulin significantly enhanced the lipofection efficiency in A549 and Calu3 cells but not in H292 cells, whereas TF showed significant lipofection efficiency-enhancing effect in Calu3 and H292 cells but not in A549 cells. The transfection efficiency correlated well with the amounts of DNA delivered to the nucleus as well as the amounts of the receptor. These results indicate that the gene delivery strategy employing ligand-facilitated lipofection can achieve high transfection efficiency in human lung carcinoma cells. In addition, enhancement of the expression of the receptor may be a possible strategy for increasing the efficiency of gene targeting.

Cell Nucleus↗
PubMed2000

Value of intra-arterial calcium stimulated venous sampling for regionalization of pancreatic insulinomas.

BACKGROUND: Intra-arterial calcium stimulation with hepatic venous sampling (ASVS) for insulin gradients has been reported to be the most sensitive preoperative localizing technique for insulinomas. We reviewed our experience with ASVS to localize and guide the treatment of insulinomas over the past decade. METHODS: Eighteen patients who underwent ASVS before surgical exploration for insulinoma were studied. The accuracy of ASVS was compared with intraoperative findings and other localizing studies. RESULTS: There were no complications arising from the procedures. A more than 2-fold step-up in insulin level 30 to 60 seconds after injection to at least 1 feeding artery was observed in 16 patients. Fourteen of the 16 solitary tumors (87.5%) were correctly located; 100% (6/6 tumors) at the head and 80% (8/10 tumors) at the body/tail. The overall accuracy of this test was 89%, compared with 11%, 33%, 38%, and 63% of ultrasonography, computed tomography, magnetic resonance imaging, and endoscopic ultrasonography, respectively. Six enucleations and 10 distal resections were performed, which included 2 laparoscopic procedures. The combination of intraoperative ultrasonography with preoperative ASVS identified all tumors. CONCLUSIONS: ASVS is the most accurate preoperative localization tool for the localization of insulinomas and, in combination with intraoperative ultrasonography, can enhance surgical success.

Adult↗
PubMed1999

A case-controlled MRI/MRA study of neurovascular contact in hemifacial spasm.

BACKGROUND: Neurovascular contact (NVC) with the root exit zone (REZ) of the ipsilateral facial nerve is associated with hemifacial spasm (HFS), but unresolved issues remain. OBJECTIVES: To 1) determine the frequency of symptomatic and nonsymptomatic NVC, 2) determine the features of NVC associated with HFS, and 3) correlate severity of HFS to these features. METHODS: Two independent, blinded, prospective assessments of high-resolution MR and MR angiography (MRA) images were performed on Chinese cases (HFS: n = 44; age-matched control subjects: n = 20). RESULTS: Over 88% of 44 symptomatic sides in patients with HFS had NVC of the ipsilateral facial nerve. At least 80% of symptomatic sides involved NVC at the anterior aspect of the REZ [REZ(ant.)]. Although NVC was observed in approximately half of nonsymptomatic sides, at least 70% of them were not at REZ(ant.). NVC at the cisternal and intracanalicular portions of the facial nerve were not associated with HFS. Half of our patients with HFS had bilateral NVC, but none had bilateral symptoms. Most of our MR/MRA images showed that the size and position of the arterial branches of the vertebrobasilar system were markedly asymmetric. Of patients with bilateral NVC, over 83% had asymmetric NVC sites. The anterior inferior cerebellar artery was the most common vessel involved in NVC, but was not significantly associated with HFS. Most of the NVC involved one vessel at one contact point with no indentation. The development of HFS was significantly associated with nerve indentation in NVC. The development and severity of HFS were not associated with multiple contact points in NVC. No significant interobserver variability existed between the blinded assessments. CONCLUSIONS: MRI/MR angiography are accurate, fast, and safe in characterizing neurovascular contact (NVC) at the brainstem. The site of NVC and ipsilateral facial nerve indentation in NVC are significant determinants for the development of hemifacial spasm (HFS). The lack of bilateral NVC at the anterior aspect of the root exit zone of the facial nerve could explain in part the lack of bilateral symptoms. The development and severity of HFS are not associated with a specific blood vessel or multiple contact points in NVC.

Adult↗
PubMed1999

Both ATP sites of human P-glycoprotein are essential but not symmetric.

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Lectin enhancement of the lipofection efficiency in human lung carcinoma cells.

Poor transfection efficiency of human lung carcinoma cells by lipofection begs further development of more efficient gene delivery strategies. The purpose of this study was to determine whether lectins can improve the lipofection efficiency in lung carcinoma cells. A549, Calu3, and H292 cells grown to 90% confluence were transfected for 18 h with a plasmid DNA containing a beta-galactosidase reporter gene (pCMVlacZ) using lipofectin plus a lectin as the vector. Ten different lectins, which exhibit a wide range of carbohydrate-binding specificities, were examined for their abilities to enhance the efficiency of lipofection. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (units/microg protein) and % blue cells following X-Gal stain. Lipofectin supplemented with Griffonia simplicifolia-I (GS-I) yields largest enhancement of the lipofection efficiency in A549 and Calu3 cells (5.3- and 28-fold, respectively). Maackia amurensis gives the largest enhancement (6.5-fold) of lipofection efficiency in H292 cells. The transfection efficiency correlates with the amounts of DNA delivered to the nucleus. Binding of FITC-labeled GS-I and the enhancement of the lipofection efficiency by GS-I were inhibited by alpha-methyl-D-galactopyranoside, indicating an alpha-galactoside-mediated gene transfer to lung carcinoma cells. We conclude that lectin-facilitated lipofection is an efficient gene delivery strategy. Employment of cell type-specific lectins may allow for efficient cell type-specific gene targeting.

Humans↗
PubMed1999

Expression of core 2 beta-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes.

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.

Amino Acid Sequence↗
PubMed1999

Development of monoclonal antibodies against bovine mucin core 2 beta6 N-acetylglucosaminyltransferase.

Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 beta6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M(-1)) for these MAbs range from 5.4 x 10(7) to 1.2 x 10(9). These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, these antibodies should be useful for further study of bovine and human core 2 enzymes.

Animals↗
PubMed1999

Patulous eustachian tube in long-term survivors of nasopharyngeal carcinoma.

This article reports on 21 long-term (10 years) survivors of nasopharyngeal carcinoma, divided into 2 groups: those subjected to an inflation-deflation test and a clearance function test in a longitudinal study, and those receiving sonotubometry in a cross-sectional study. On the inflation-deflation test, 12 (55%) out of 22 ears had a patulous eustachian tube, and on sonotubometry, 10 (50%) out of 20 ears also revealed a patulous eustachian tube. Except for 4 ears with chronic otitis media, the ears had resolved to a normal eardrum appearance at 10 years postirradiation. The phenomenon might be attributed to both restoration of the impaired tubal function and the development of a patulous tube.

Case-Control Studies↗
PubMed1998

Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells.

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were Increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.

Adenocarcinoma↗
PubMed1998

Mucin biosynthesis: molecular cloning and expression of bovine lung mucin core 2 N-acetylglucosaminyltransferase cDNA.

A cDNA clone containing a 2,150-bp insert was isolated from a bovine lung lambdagt10 cDNA library by cross-species hybridization using a DNA probe generated by polymerase chain reaction (PCR) employing a human cDNA that encodes mucin core 2 beta6-N-acetylglucosaminyltransferase (hC2TF) as the template. The bovine cDNA (bcDNA) insert was devoid of 220 bp of the 5' portion of the C2TF open reading frame (ORF), as predicted from the human counterpart. Southern blotting analysis suggested that the coding region of this C2TF gene is in one exon. To construct a full-length bovine C2TF (bC2TF) cDNA, a genomic DNA fragment containing the 5' portion of the ORF of the bC2TF gene was cloned from a lambdaEMBL bovine genomic DNA library and ligated to the 5' end of the cloned cDNA insert. DNA sequence analysis showed that the complete ORF of bC2TF gene was 1,281 bp in length, which corresponds to a polypeptide of 427 amino acids. Catalytically active bC2TF was expressed in sf21 insect cells infected with recombinant baculovirus containing the ORF of the bC2TF gene. The recombinant bC2TF catalyzed the synthesis of core 2, but not core 4 and blood group I structures. Western blotting analysis showed that the recombinant bC2TF migrated with the same mobility (approximately 55 kD) as the native bovine tracheal C2TF. Immunohistochemical analysis showed that in bovine trachea, the bC2TF was present at the surface epithelium and in the submucosal glands, with the latter being the major site of distribution.

Amino Acid Sequence↗
PubMed1997

A 10-year longitudinal study of tubal function in patients with nasopharyngeal carcinoma after irradiation.

OBJECTIVE: To follow up the chronological change of tubal function in patients with nasopharyngeal carcinoma for 10 years after irradiation. DESIGN: Patients with nasopharyngeal carcinoma were subjected to eustachian tube function tests before irradiation and 6 months, 5 years, and 10 years after irradiation. SETTING: University hospital. PATIENTS: Ten patients (20 ears), 7 men and 3 women. RESULTS: Twelve (60%) of the ears had patulous tubes 10 years after irradiation, which might result from atrophy of peritubal tissues. The occurrence of a patulous tube was independent of the radiation dosage, but it was related to the interval since irradiation. CONCLUSIONS: Development of a patulous tube is associated with correction of organic obstruction of the eustachian tube. Resolution of the inflammatory reaction plays the most important role in recovery of function of the tube. Therefore, the prevalence of middle ear complications is low 10 years after irradiation.

Adult↗
PubMed1997

Regulation of mucin secretion in the ferret trachea.

Mucin is the major component of mucus and can be used as a marker for mucus secretion. The purpose of this study was to develop an in vitro method to evaluate the regulation of mucin secretion. To do this, we used a sandwiched enzyme-linked lectin assay to measure mucin secretion from isolated ferret tracheal segments. This assay entailed coating microtiter plate wells with dolichos biflorus agglutinin and detecting the bound mucin that was secreted into a buffer solution by the tracheal segments. We used this method to evaluate the secretory response to four secretagogues: prostaglandin F2alpha (PGF2alpha), adenosine triphosphate (ATP), methacholine, and human neutrophil elastase (HNE). Each agent stimulated mucin secretion above baseline secretion (ATP (p = 0.022), PGF2alpha (p = 0.009), and HNE (p < 0.05)), and the relative potency of these secretagogues was PGF2alpha < or = ATP < MCh < HNE. We also demonstrated that there is an anatomic gradient for both constitutive and stimulated mucin secretion, with the distal tracheal segments secreting more mucin per gram of weight than the proximal segments. This fairly simple and reproducible technique can be used to evaluate the regulation of mucin secretion in the airway and to assess the efficacy of agents that might alter the secretory response.

Adenosine Triphosphate↗
PubMed1997

Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis.

Cytokine levels in nasal and lower airways in young cystic fibrosis (CF) patients were compared with those in controls. Nasal (NLF) and bronchoalveolar (BALF) lavage fluids were obtained from children with or without CF who were undergoing bronchoscopy for clinical indications. In NLF, neither inflammatory cells nor cytokine concentrations differed between patients and controls. However, interleukin (IL)-8 levels in infected BALF from children with CF were markedly elevated compared with levels in infected and uninfected controls, even after standardization of IL-8 concentrations to bacterial counts. BALF IL-6 was modestly elevated in infected CF patients compared with uninfected but not infected controls; IL-10 did not differ among the groups. NLF and BALF IL-8 levels were not significantly correlated. Excessive airway inflammation in early CF thus appears to be confined to the lower respiratory tract, and IL-8 levels are markedly increased in children with CF compared with control children with a bacterial infection of the lower airways.

Bacterial Infections↗
PubMed1997

Bilateral Kienbock's disease in SLE.

Kienbock's disease, or radiologic avascular necrosis (AVN) of the carpal lunate, is an uncommon disease of young adults. The precise etiological mechanisms are uncertain although multiple mechanical, anatomical, and vascular factors are thought to be involved. A patient with systemic lupus erythematosus (SLE) who developed bilateral Kienbock's disease during the course of her illness was reported. A vasculopathy related to active vasculitis or the antiphospholipid antibodies was thought to be responsible.

Adult↗