Biomedical subjects

J L Su

Publications and source records attributed to J L Su.

At least 19 recordsLinked to original sources

PubMed2006

Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments.

OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.

ADAM Proteins↗

PubMed2001

The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K/Akt pathway.

Interleukin-6 (IL-6), a multifunctional cytokine, has recently been implicated in human cervical cancer, though the mechanism remains elusive. This study demonstrates that the anti-apoptotic protein Mcl-1 and IL-6 was concomitantly expressed in human cervical cancer tissues and cell lines, but not in normal cervix tissues. Upon IL-6 treatment, Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated peaking at 4-8 h in human cervical cancer C33A cells. Supporting this observation, using anti-IL-6 or anti-IL-6 receptor antibody to interrupt the IL-6 autocrine loop in SiHa cells significantly reduced cellular level of Mcl-1. This study hypothesizes that the expression of Mcl-1 in cervical cancer cells is regulated by IL-6. The matter of which signaling pathways transduced by IL-6 is responsible for the Mcl-1 up-regulation is further investigated herein. Blocking the STAT3 or MAPK pathway with dominant-negative mutant STAT3F or the MEK inhibitor PD98059 failed to inhibit IL-6-mediated Mcl-1 expression. Meanwhile, the IL-6-induced Mcl-1 up-regulation was effectively abolished by treatment with PI 3-K inhibitors, LY294002. Additionally, overexpression of dominant-negative (dn) Akt in C33A cells could inhibit the IL-6-induced increase of Mcl-1. Finally, overexpression of IL-6 in C33A cells caused a markable resistance to apoptosis induced by doxorubicin or cisplatin. Transient transfection of IL-6-overexpressed cells with a mcl-1 antisense vector, leading to the attenuation of their apoptosis-resistant activity. In conclusion, the data herein suggest that IL-6 regulated the mcl-1 expression via a PI 3-K/Akt-dependent pathway that may facilitate the oncogenesis of human cervical cancer by modulating the apoptosis threshold.

Antineoplastic Agents↗

PubMed2001

Progression of symptoms in the early and middle stages of Huntington disease.

OBJECTIVE: To delineate the progression of symptoms in the early and middle stages of Huntington disease (HD). DESIGN: A survey of individuals with symptomatic HD completed by a first-degree relative. SETTING: The National Huntington Disease Research Roster for Patients and Families, Indianapolis, Ind. PARTICIPANTS: The survey included 1238 individuals with a minimum of a 6-year history of symptomatic HD. MEASURES: Participating families completed a series of surveys, including the Affected Individual Questionnaire, which consists of 19 physical, emotional, and cognitive signs commonly thought to occur during disease progression. The respondent indicates if each of the symptoms occurred and, if so, at what time during the course of the disease: (1) within 1 year, (2) within 2 to 5 years, (3) within 6 to 10 years, (4) after more than 10 years, (5) has not occurred, or (6) "don't know." RESULTS: The symptoms are categorized into 6 onset periods. Involuntary movements are grouped alone as the earliest reported symptom. The second group is composed entirely of mental and emotional symptoms, including sadness, depression, and difficult to get along with. The third group includes clumsiness, sexual problems, lack of motivation, and suspiciousness/paranoia. As the disease progresses, a variety of motor, emotional/behavioral, and cognitive symptoms are experienced, including unsteadiness, trouble holding onto things, trouble walking, changes in sleeping patterns, delusions and hallucinations, intellectual decline, and memory loss. With the approach of late-stage HD, affected individuals begin to experience speech difficulty and weight loss. In the late stage, patients lose bowel and bladder control. CONCLUSIONS: Even though the symptoms of HD are fairly well characterized, their progression, especially in the early and middle stages, remains uncertain. Clarification of the disease progression is vital to improved understanding of the pathogenesis of HD and to the evaluation of therapeutic agents that are designed to slow the progression of disease. The results of this study assist in clarifying HD progression from early involuntary movements and emotional changes to more overt motor symptoms and difficulty with activities of daily living.

Activities of Daily Living↗

PubMed2001

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Animals↗

PubMed2000

Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.

Animals↗

PubMed2000

Suramin analogs inhibit human angiogenesis in vitro.

BACKGROUND: Suramin is a polysulfonated naphthylurea that inhibits tumor cell proliferation and angiogenesis, but the widespread use of this drug has been limited by significant neurologic toxicity. A series of suramin analogs that may exhibit less toxicity in vivo have been synthesized. We hypothesized that these novel analogs would have antiangiogenic properties equal to or greater than those of suramin when evaluated in an in vitro human placental vein angiogenesis model. METHODS: Human placental veins (n = 72 per group) were cultured in a 0.3% fibrin clot for a period of 14 days. Three suramin analogs (NF 145, NF 248, NF 293) and suramin were tested at 56 and 560 microM concentrations to determine their effect on the development of an angiogenic response. Experiments were repeated for each analog on veins from three different placentas. The percentage of wells that initiated an angiogenic response was calculated and compared with initiation in a control group (n = 141). RESULTS: The three suramin analogs inhibited angiogenesis in a dose-dependent fashion, with all compounds exhibiting near-complete inhibition of angiogenesis at 560 microM. The effects of these analogs were equal to or greater than those of suramin. CONCLUSION: Suramin analogs with structural alterations inhibit human angiogenesis at concentrations equivalent to those seen in vivo. These analogs may be more effective antiangiogenic agents than suramin and may have less potential for toxicity.

Animals↗

PubMed2000

Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.

Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.

Animals↗

PubMed2000

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Acid Anhydride Hydrolases↗

PubMed2000

Production and characterization of an estrogen receptor beta subtype-specific mouse monoclonal antibody.

An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expression pattern of each of these receptors. We report the generation and characterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is ERbeta subtype-specific and capable of recognizing full-length human ERbeta as well as all of its known protein isoforms. ER15.64A, raised against a ERbeta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to the immunizing peptide and the full-length E. coli expressed ERbeta in ELISA and BIAcore assays. It also immunostained nuclei of Sf9 insect cells that were infected with an ERbeta-baculovirus. In Western analysis, ER15.64A recognized ERbeta1 and ERbeta2 proteins from a reticulocyte in vitro transcription/translation preparation. This antibody did not cross-react with recombinant ERalpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis. The specificity of ER15.64A should make this antibody a useful tool for monitoring expression of ERbeta and its isoforms at the protein level and should aid in distinguishing the pattern of ERbeta receptor expression from that of ERalpha.

Animals↗

PubMed2000

Endoscopic management of skull base osteoradionecrosis.

OBJECTIVE: Osteoradionecrosis is one of the most serious complications in radiotherapy of nasopharyngeal carcinoma. We describe a new endoscopic approach to resolve resultant skull base osteoradionecrosis. The objective of this study is to evaluate the efficacy of endoscopic management of skull base osteoradionecrosis. STUDY DESIGN: A prospective study of the outcome of endoscopic management for patients with skull base osteoradionecrosis. METHODS: Between 1994 and 1998 six patients who had irradiation previously for nasopharyngeal carcinoma had skull base osteoradionecrosis. A sinoscopic approach was applied for diagnosis and sequestrectomy. This diagnosis was based on the criterion of exposed necrotic bone after removing all crust in the nasopharynx and further confirmed on pathological examination after sequestrectomy. Effective cure was defined as intact mucosal coverage without any ulcer or exposed necrotic bone observed in the nasopharynx and the absence of antecedent accompanying symptoms after management. RESULTS: Six patients (10%) were symptom free. Five (83.3%) patients had effective cure. There was no surgical morbidity or mortality. CONCLUSION: Endoscopic sequestrectomy is a justified approach to skull base osteoradionecrosis.

Adult↗

PubMed1999

Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer.

It has been shown in previous studies that a variety of estrogen receptor (ER) beta mRNA transcripts are expressed in human breast cancer cell lines and tumors. To complement the RNA expression studies, we have developed ER-beta-specific antibodies to characterize ER-beta protein expression in breast cancer cell lines and tumors. Monoclonal antibodies were made against a peptide representing the first 18 amino acids of the longest ER-beta open reading frame reported to date, and polyclonal antibodies were made against a peptide within the ER-beta B domain. By Western blot analysis, we show that ER-beta protein is expressed in all cancer cell lines tested and in three of five breast tumor samples. The breast cancer cell lines showed variation in the size of the expressed ER-beta protein. The longest form detected was consistent with the 530-amino acid, full-length ER-beta sequence. Shorter ER-beta isoforms were detected in the ER-alpha-negative MDA-MB-231 and MDA-MB-435 breast cancer cell lines, likely corresponding to previously described COOH-terminal RNA variant isoforms.

Blotting, Western↗

PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗

PubMed1999

Use of a PPAR gamma-specific monoclonal antibody to demonstrate thiazolidinediones induce PPAR gamma receptor expression in vitro.

Troglitazone and rosiglitazone (BRL49653), members of the thiazolidinedione (TZD) class of antidiabetic drugs, are peroxisome proliferator-activated receptor gamma (PPARgamma) ligands that induce adipocyte differentiation and increase the expression of PPARgamma protein. Here, we report the characterization of a PPARgamma specific monoclonal antibody (MAb), PgammaA53.25, and its use to monitor PPARgamma expression in the noncommitted pluripotent murine mesenchymal stem cell line, C3H10T1/2, treated with TZDs. MAb PgammaA53.25 was raised against a region in the N-terminal domain of human PPARgamma shared by splice variants PPARgamma1 and PPARgamma2. It recognizes immunizing antigen in enzyme-linked immunoadsorbent assay (ELISA), and does not cross-react with the N-terminal domains of PPARalpha or PPARdelta. In Western blotting, PgammaA53.25 reacts with the immunizing antigen as well as distinct protein bands corresponding to the molecular weight of full length PPARgamma from C3H10T1/2 cells and rat tissue lysates. In fluorescent microscopy, PgammaA53.25 immunostains nuclei of C3H10T1/2 cells treated with PPARgamma ligands. The fluorescence intensity of the treated cells is TZD dose-dependent, and correlates with lipid accumulation consistent with adipogenesis. Based on these results, we propose that MAb PgammaA53.25 will be a useful tool for elucidating the role of PPARgamma in fatty acid metabolism and adipocyte differentiation.

Adipocytes↗

PubMed1999

Systematic management of osteoradionecrosis in the head and neck.

OBJECTIVES: Osteoradionecrosis is one of the most serious and devastating complications of radiotherapy. The proper management of osteoradionecrosis is currently undetermined. The objective of this study is to evaluate the treatment results of a systematic approach to osteoradionecrosis. STUDY DESIGN: A prospective study of a systematic approach to osteoradionecrosis in the head and neck area was undertaken. METHODS: From July 1993 to June 1998, 33 cases of osteoradionecrosis in the head and neck area were treated using a systematic approach that combined sequestrectomy and hyperbaric oxygen therapy. RESULTS: Seven (21%) had recurrent cancer. The control rate of the other 26 osteoradionecrosis cases was 77% (20/26). CONCLUSIONS: Persistent osteoradionecrosis, despite diligent radical treatment, raises the suspicion of recurrent cancer. Extensive osteoradionecrosis with a multiple discharging fistula, a large area of exposed necrotic bone, or a coexistent fracture should be treated primarily with radical sequestrectomy and microvascular free flap reconstruction. Surgery still plays a major role in controlling osteoradionecrosis, and hyperbaric oxygen therapy is adjuvant.

Bone Diseases↗

PubMed1999

[Effects of myocardial ischemia reperfusion injury on L-arginine/nitric oxide system in rat heart].

Ischemia reperfusion injury (IRI) model of rat heart was prepared by preperfusion for 15 min, then a suspension for 45 min and recycling reperfusion for 15 min with 30 ml KH buffer. The leakage of lactate dehydrogenase(LDH),protein, myoglobin and nitrite (NO2-) in the circular perfusion fluid were measured. Myocardial nitric oxide synthase(NOS) activity and L-arginine transport were observed. In the IR group, the leakage of LDH,protein, myoglobin and NO2- were increased respectively by 4.1,5.4,1 and 1.2 times(P<0.01) and NOS(tNOS, iNOS, cNOS) activity by 48.2%,43.2% and 52.1%,(P<0.01,respectively) as compared with the control group. L-arginine transport might be mediated by either high- or low-affinity transport system in cardiac tissue. In the IR group, L-arginine transport increased significantly with the V(max) being increased by 48% and 2 times respectively for the low-affinity and the high-affinity transport as compared with control. Michaelis constant (km) was decreased by 47.4% for low-affinity transport (P<0.05),but not significantly changed for the high-affinity transport. These results suggest that the increase of nitric oxide generation might result from the increased myocardial NOS activity and L-arginine transport during IRI.

Animals↗

PubMed1998

Cloning and characterization of human estrogen receptor beta isoforms.

Multiple transcripts which arise from the human estrogen receptor beta (ER beta) gene have been characterized. Three full length isoforms of the hER beta gene, designated hER beta 1-3, were identified in a testis cDNA library. An additional two isoforms, designated hER beta 4 and hER beta 5, were identified by PCR amplification from testis cDNA and from the MDA-MB 435 cell line. hER beta 1 corresponds to the previously described hER beta. All five isoforms diverge at a common position within the predicted helix 10 of the ligand binding domain of hER beta, with nucleotide sequences consistent with differential exon usage. The hER beta isoform mRNAs displayed a differential pattern of expression in human tissues and in tumor cell lines when analyzed by RT-PCR. Further characterization of the three full length isoforms, hER beta 1-3, by in vitro band shift studies indicated that the isoforms were able to form DNA-binding homodimers and heterodimers with each other and with the ER alpha subtype.

Alternative Splicing↗

PubMed1998

Monitoring of PPAR alpha protein expression in human tissue by the use of PPAR alpha-specific MAbs.

We report the production and characterization of two PPAR alpha subtype-specific monoclonal antibodies raised against the N-terminal domain of PPAR alpha. P alpha b 11.80A is a Western-reactive antibody, whereas P alpha b 32.51 is useful for immunohistochemistry. Both antibodies exhibited high affinity against the immunogen based on BIAcore analysis, recognized full-length PPAR alpha protein in PPAR alpha-transfected CV-1 cells, and displayed no cross-reactivity against the N-terminal domains of PPAR gamma or PPAR delta proteins as demonstrated by various immunoassays. The application of these antibodies to a panel of normal human tissues revealed that PPAR alpha protein expression is highest in skeletal muscle, liver, and kidney, consistent with previously reported mRNA expression data. These antibodies provide us with valuable tools to further explore the function of PPAR alpha.

Antibodies, Monoclonal↗

PubMed1998

A unified multimedia database system to support telemedicine.

A unified approach to managing multimedia medical databases in a telemedicine system is proposed. In order to manage, search, and display patient information more efficiently, we define a patient information package (PIP) as a concise data set of a patient's medical information from each visit. By means of PIP's, both patient-oriented and problem-oriented query strategies, which are most frequently used in daily clinical practice and medical education, can be accommodated. We also provide a unified methodology for accessing various types of patient medical records as well as design two types of user interfaces, high-quality data display and web-based interface, for different medical service purposes. The PIP-based management of databases has been successfully implemented between the National Taiwan University (NTUH), Taipei, and the Chinshan health care center, Chinshan, Taiwan, for teleconsultation, telediagnosis, and tele-education.

Database Management Systems↗