Biomedical subjects

J Lin

Publications and source records attributed to J Lin.

At least 361 records · Page 20Linked to original sources

PubMed1998

An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene.

Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob/ob (leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.

Adenoviridae↗

PubMed1998

Protein kinase C isoform expression and function in transformed and non-transformed pancreatic acinar cell lines.

Members of the protein kinase C (PKC) family of multifunctional serine/threonine phosphorylating enzymes are believed to play a role in regulating cellular differentiation and proliferation in many cell types. In the present study, we examined the expression of PKC isoforms in non-transformed (BMRPA.430) and transformed (TUC3) rat pancreatic acinar cell lines and compared this to PKC expression in freshly dispersed acini from rat pancreas. BMRPA.430 cells maintain characteristics of normal acini and are not tumorigenic, whereas TUC3 cells do not express tight junctions or polygonal morphology and are tumorigenic. As reported previously, PKC alpha, delta, epsilon, and zeta are expressed in freshly prepared acini. Likewise, these isoforms were detected in both the BMRPA.430 and TUC3 cell lines. In addition, PKC theta, a novel isoform, was detected in all three cell types at low levels. We used two PKC inhibitors to examine the role of PKC in acinar cell proliferation. CGP 41 251, a selective PKC inhibitor, and Go 6976, an agent which specifically inhibits calcium-dependent PKC isoforms, inhibited cell proliferation of both cell lines. Translocation of PKC alpha to the membrane was not observed in either cell line. Hence, our data indicate that ras-induced transformation does not alter PKC isoform expression in pancreatic acinar cells and that activation of PKC alpha is involved with acinar cell growth.

Animals↗

PubMed1998

Major histocompatibility class II-mediated signal transduction is regulated by the protein-tyrosine phosphatase CD45.

Major histocompatibility complex class II molecules and the B cell antigen receptor (BCR) transduce similar signals when cross-linked by ligand. Therefore, studies were conducted to determine whether the protein tyrosine phosphatase CD45 regulates signaling via these transmembrane receptors in an analogous manner. Cross-linking of either class II molecules or the BCR on CD45-positive K46-17micromlambda B lymphoma cells was observed to induce activation of the Src family protein- tyrosine kinase Lyn, tyrosine phosphorylation of Syk and phospholipase Cgamma, and the production of inositol 1,4,5-trisphosphate leading to intracellular mobilization as well as extracellular influx of Ca2+. In the absence of CD45, cross-linking of either class II molecules or the BCR failed to induce activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a normal manner, whereas phospholipase Cgamma exhibited a high basal level of tyrosine phosphorylation that was not significantly increased upon stimulation. Nevertheless, phospholipase Cgamma appeared to be functional because CD45-negative cells produced elevated levels of inositol 1,4,5-trisphosphate following stimulation through class II or the BCR. Regardless of this, CD45-negative cells exhibited Ca2+ mobilization responses that were greatly diminished and transient in nature. Whereas little or no mobilization of Ca2+ was observed in response to class II cross-linking, CD45-deficient cells mobilized Ca2+ from intracellular stores but not the extracellular environment in response to BCR cross-linking. These results demonstrate that CD45 regulates both Src family kinase activation and Ca2+ mobilization associated with class II- and BCR-mediated signal transduction.

Animals↗

PubMed1998

Angiostatin upregulates E-selectin in proliferating endothelial cells.

Angiostatin, a 38 kilodalton fragment of plasminogen, is a potent inhibitor of angiogenesis. However, little is known about how angiostatin affects endothelial gene expression. To learn more about its effect on endothelial-specific genes implicated in angiogenesis, we examined E-selectin expression and function in bovine capillary endothelial cells treated with recombinant angiostatin. Angiostatin caused a four to five-fold increase in E-selection polypeptide levels in proliferating endothelial cells but little or no increase in confluent cells. P-selection polypeptide levels were unaffected by angiostatin in either proliferating or confluent cells. E-selectin mRNA and adhesion activity in proliferating endothelial cells were also increased by angiostatin. Angiostatin had little effect on the distribution of endothelial cells in G0/G1, S, and G2/M, indicating angiostatin does not alter cell cycle progression significantly. These data demonstrate that angiostatin selectively upregulates E-selectin in proliferating endothelial cells in vitro. This selectivity may provide insights into the mechanism by which angiostatin inhibits tumor growth in vivo without apparent effects on quiescent endothelium.

Angiostatins↗

PubMed1998

Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength.

The reaction between metmyoglobin and imidazole has been studied as a function of pH between pH 4.2 and 11.5 and as a function of ionic strength at integral pH values (5 to 10) between 0.001 and 1.0 M ionic strength. The reaction between metmyoglobin and 1-methylimidazole has also been investigated as a function of pH. Comparison of the pH dependence of the association rate constants for the two ligands indicates that the negatively charged imidazolate ion does not contribute to the observed rate of imidazole binding at pH < or = 11.5. At all pH values between pH 4.2 and pH 11.5 the initial complex formed involves the neutral form of bound imidazole. At pH 11.5, the neutral imidazole complex is converted slowly (t1/2 approximately 10 s) into an imidazolate complex. The kinetic data were analyzed according to two mechanisms, one involving the binding of neutral imidazole only and one involving the direct binding of both imidazole and the imidazolium ion to metmyoglobin. Although secondary kinetic salt effects account for the ionic strength dependence of the association rate constant, evidence which indicates that metmyoglobin reacts with imidazole and with the imidazolium ion with similar rates is provided. A self-consistent analysis indicates that the rate constants for imidazole and imidazolium ion binding to metmyoglobin are 350 and 230 M-1 s-1, respectively, at neutral pH and 0.1 M ionic strength. Imidazole can react directly with hydroxymetmyoglobin with a rate of 56 M-1 s-1 at 0.1 M ionic strength, about sixfold slower than binding to aquometmyoglobin. Protonation of a second heme-linked group, thought to be His-97, has little influence on the binding of imidazole but does decrease the rate of imidazolium binding by about eightfold to 29 M-1 s-1 at 0.1 M ionic strength.

Animals↗

PubMed1998

Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.

Human skin is exposed daily to solar ultraviolet (UV) radiation. UV induces the matrix metalloproteinases collagenase, 92-kD gelatinase, and stromelysin, which degrade skin connective tissue and may contribute to premature skin aging (photoaging). Pretreatment of skin with all-trans retinoic acid (tRA) inhibits UV induction of matrix metalloproteinases. We investigated upstream signal transduction pathways and the mechanism of tRA inhibition of UV induction of matrix metalloproteinases in human skin in vivo. Exposure of human skin in vivo to low doses of UV activated EGF receptors, the GTP-binding regulatory protein p21Ras, and stimulated mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38. Both JNK and p38 phosphorylated, and thereby activated transcription factors c-Jun and activating transcription factor 2 (ATF-2), which bound to the c-Jun promoter and upregulated c-Jun gene expression. Elevated c-Jun, in association with constitutively expressed c-Fos, formed increased levels of transcription factor activator protein (AP) 1, which is required for transcription of matrix metalloproteinases. Pretreatment of human skin with tRA inhibited UV induction of c-Jun protein and, consequently, AP-1. c-Jun protein inhibition occurred via a posttranscriptional mechanism, since tRA did not inhibit UV induction of c-Jun mRNA. These data demonstrate, for the first time, activation of MAP kinase pathways in humans in vivo, and reveal a novel posttranscriptional mechanism by which tRA antagonizes UV activation of AP-1 by inhibiting c-Jun protein induction. Inhibition of c-Jun induction likely contributes to the previously reported prevention by tRA of UV induction of AP-1-regulated matrix-degrading metalloproteinases in human skin.

Activating Transcription Factor 2↗

PubMed1998

Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium.

Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E. coli O157-associated hemorrhagic colitis. We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-1 (ET-1) and nitric oxide (NO). In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h. Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis. In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged. Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-[14C]arginine to L-[14C]citrulline conversion or the NO chemiluminescence signal. The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations. Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells. Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis. Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the microvascular dysfunction that is the hallmark of hemolytic uremic syndrome and hemorrhagic colitis.

Animals↗

PubMed1998

Lineage analysis using retroviral vectors.

Knowledge of the genealogical relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Genealogical relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. The use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues is described. An overview of the relevant aspects of the retroviral life cycle is given, and the strategies and current methods in use in our laboratory are described.

Animals↗

PubMed1998

Bilateral testicular tumors in an infant.

A 7-month-old infant showed bilateral enlarged, nontender scrotal masses. The level of alpha-fetoprotein was greater than 10,000 ng/ml preoperatively; a high left inguinal orchiectomy was performed for a suspected yolk-sac tumor. The right testis was diagnosed as a mature teratoma because it was not possible to establish a line of cleavage between the tumor and the normal tissue, and a high right inguinal orchiectomy was performed. Only one case of bilateral testicular teratomas has been reported in the literature to date. We report a rare second case of bilateral testicular tumors, one a yolk-sac tumor and the other a teratoma.

Endodermal Sinus Tumor↗

PubMed1998

Control of effective expression of the phage phiCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site.

In the DNA of bacteriophage phiCTX, the attachment site (attP), the cohesive end site (cos), and the gene (ctx) encoding the pore-forming cytotoxin, are clustered. Deletion variants and PCR-generated fragments of the DNA were cloned into the Pseudomonas aeruginosa and Escherichia coli vector pHA10. Recombinant plasmids carrying the chloramphenicol acetyltransferase gene, cat, were used for promoter studies. Two promoters responsible for ctx expression are located between attP and cos and between cos and ctx, the promoter upstream of cos being stronger than the one downstream. The promoters and the ribosomal binding site are recognized by both the P. aeruginosa and E. coli transcriptional systems. The results indicate that chromosomal integration of phiCTX DNA, which requires cos site ligation, is necessary for high ctx expression in phiCTX-infected P. aeruginosa strains.

Base Sequence↗

PubMed1998

Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet.

Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.

Bacteriophage lambda↗

PubMed1998

Molecular cloning of genes differentially regulated by TNF-alpha in bovine aortic endothelial cells, fibroblasts and smooth muscle cells.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic-cytokine binding to and thereby stimulating vascular cells. TNF-alpha mediated intermediate stimulation of vascular cells is believed to play a pivotal role in the development of arteriosclerosis. While extensive information has recently become available on gene induction by TNF-alpha, less is known about gene suppression by TNF-alpha in vascular cells. Endothelial cells are the first cell layer within the vessel wall interacting with circulating, cytokine releasing cells. Therefore, they were selected as target for these study. METHODS: A differential screening approach has been used to isolate cDNAs whose abundance was suppressed by incubating bovine aortic endothelial cells (BAEC) for 6 h with 1 nM TNF-alpha. The gene expression of 6 isolated cDNAs after TNF-alpha was investigated by dot blots and nuclear run-on analysis in BAEC. The investigated genes were partially or completely sequenced. Differential expression after TNF-alpha stimulation of BAEC, bovine fibroblasts and vascular smooth muscle cells (SMC) was studied by Northern blots. RNA transcripts of the clone C7 in aortic aneurysms were examined by in situ hybridization. RESULTS: 49 independent cDNAs were isolated by the differential screening approach and 6 clones were further analyzed. These genes were downregulated in a time and dose dependent manner in BAEC. Sequence analysis revealed that 3 cDNAs encoded previously unidentified genes (C1, C5, C7), while 3 encoded known genes: connective tissue growth factor (CTGF; A1), fibronectin (A8) and the mitochondrial genome (B1). A1 and B1 were suppressed in BAEC, fibroblasts and SMC, whereas A8, C1, C5 and C7 were not uniformly downregulated in the investigated cells. C7 RNA transcripts were exclusively induced in the endothelium of an uninflamed aortic aneurysm. The transcripts were undetectable in an inflamed aortic aneurysm and control vessels. CONCLUSIONS: Gene suppression is a prominent feature of the intermediate effect of TNF-alpha on endothelial cells. Differences in the expression of the tested genes in endothelial cells, fibroblasts and vascular smooth muscle cells open possibilities for the study of cellular interactions in the vascular wall in disease situations with high local TNF-alpha concentrations.

Animals↗

PubMed1998

Repeated exposure to trimethylolpropane phosphate induces central nervous system sensitization and facilitates electrical kindling.

Trimethylolpropane phosphate (TMPP), pentylenetetrazol (PTZ) and N-methyl-beta-carboline-3-carboxamide (FG-7142) were evaluated and compared for facilitation of electrical kindling in freely moving rats. Stimulating/recording electrodes were implanted in the left amygdala (LAD), right amygdala (RAD) and left bed nucleus (LBN) of the stria terminalis. TMPP (0.275 mg/kg), PTZ (20 mg/kg), FG-7142 (7.5 mg/kg) or vehicle was administered intraperitoneally (i.p.) to separate groups of rats 3 times/week for 10 weeks. Stimulation of the LAD (0.1 Hz, 0.1-ms duration, 280-1500 microA, 20 pulses) 24 h following the drug administration evoked epileptiform after-discharges (ADs) in the LBN and RAD of 12.5% and 17% of rats after the seventh dose of TMPP and PTZ, respectively, and in 20% of rats from the LBN and RAD after the ninth and nineteenth dose of FG-7142, respectively. The same stimulation also induced myoclonic jerks after nine doses of TMPP or PTZ, or after thirteen doses of FG-7142 in 25%, 30% and 20% of animals tested, respectively. Chemically kindled clonic seizures were observed in 100% of TMPP or FG-7142 and 50% of PTZ treated rats by the thirtieth dosing. Control animals exhibited neither behavioral nor electrographic seizures to vehicle injection or to the LAD stimulation. Kindling stimulation applied to the LAD (60 Hz, 2-s train duration, 20-1500 microA, 0.1-ms pulse duration) 4 weeks following the completion of drug treatments evoked epileptic after-discharges from the LAD, LBN and RAD in all treated groups, with generally decreased threshold and latency to onset of after-discharges, compared to vehicle controls. The present study suggests that repeated exposure of rats to sub-convulsive doses of TMPP, PTZ and FG-7142 induces long-term central nervous system sensitization that may be related to both chemical kindling and the facilitation of electrical kindling.

Animals↗

PubMed1998

Induction of immunomodulating cytokines by a new polysaccharide-peptide complex from culture mycelia of Lentinus edodes.

The immunomodulating effect of a new polysaccharide-peptide complex from culture mycelia of Lentinus edodes (LE) was studied for elucidation of the mechanism of augmentation of cell-mediated immunity. RNA samples were isolated from the untreated and treated murine splenocytes and human peripheral blood mononuclear cells. RT-PCR was used to analyze the cytokine gene expression and bioassay was used to analyze the cytokine production. By administration of LE, the expression levels of IL-2 and TNF-alpha genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-alpha were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-alpha were augmented in the treated human peripheral blood mononuclear cells. These results suggest that LE may induce Th immune responses.

Animals↗

PubMed1998

Production and testing of Schistosoma japonicum candidate vaccine antigens in the natural ovine host.

The objectives of this work were to clone and express Chinese strain Schistosoma japonicum antigens and evaluate their immunogenicity and protective efficacy in the natural ovine host in China. Recombinant antigens selected for testing were: isoforms of glutathione S-transferase Sj28GST and Sj26GST; the large hydrophilic domain of Sj23, the homologue of the protective S. mansoni membrane antigen Sm23; and a 3' fragment of S. japonicum paramyosin. In addition, Chinese strain S. japonicum native paramyosin and GST were purified and used for vaccination. Antigens were co-administered with Freund's adjuvants or BCG. We also examined the effects of co-administration of native unfractionated GSTs with keyhole limpet haemocyanin (KLH), which shares a cross-reactive protective epitope with schistosomes. These are the first side-by-side comparisons of candidate defined-antigen schistosomiasis vaccines in a natural host. Significant partial protection was obtained with each of the antigens tested. Less protection was obtained with a recombinant fragment of S. japonicum paramyosin compared with native paramyosin. Co-administration of native GST and KLH was no more effective than vaccination with either antigen alone. Although encouraging levels of protection against S. japonicum were demonstrated using each of these antigens, further work is needed to optimise vaccine delivery and vaccination schedules.

Adjuvants, Immunologic↗

PubMed1998

A 4-year study of plasma ochratoxin A in a selected population in Tokyo by immunoassay and immunoaffinity column-linked HPLC.

Employing a competitive ELISA (cELISA) based on monoclonal antibody and a novel immunoaffinity-column (IAC)-linked HPLC-fluorometry, ochratoxin A (OTA) levels in the plasma of 184 healthy volunteers (130 males, 54 females) were surveyed in Tokyo during the 4 years from 1992 to 1996. It was found that 85% of the cases were positive for OTA except 38% in 1994, and an average value in the positives was estimated as 68 pg/ml. This suggests that the population in Tokyo is exposed to OTA at high frequency, although the level in plasma is far less than that reported in Europe and Canada. A partial contribution of fermented dietary foodstuffs, coffee and wine is suspected as the source of the OTA observed in the human plasma.

Chromatography, Affinity↗

PubMed1998

Determination of the structure of oligosaccharides prepared from acharan sulfate.

The fine structure of acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica , was examined. This glycosaminoglycan has a major disaccharide repeating unit of -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1--> (where GlcNpAc is N -acetylglucosamine, IdoAp is iduronic acid, and S is sulfate) making it structurally related to both heparin and heparan sulfate. Using heparin lyases prepared from Flavobacterium heparinum and a newly isolated heparinase from Bacteroides stercoris , the controlled enzymatic depolymerization of acharan sulfate was undertaken to prepare a mixture of oligosaccharides. Fractionation of this mixture of oligosaccharides by strong-anion-exchange high performance liquid chromatography afforded oligosaccharides that capillary electrophoresis established were sufficiently pure for structural characterization. Electrospray ionization mass spectrometry identified two series of oligosaccharides, one derived from acharan sulfate's major repeating unit and a second minor group of undersulfated oligosaccharides. Proton nuclear magnetic resonance spectroscopy established the structure of these two classes of oligosaccharides to be DeltaUAp2S(1-->[4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S (1-->]n4)- D-GlcNpAcalpha,beta (where n = 0,1,2,3 and DeltaUAp is 4-deoxy-alpha-L- threo -hex-4-enopyranosyluronic acid) and DeltaUAp(1-->[4)- alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1-->]m-D-GlcNpAcal pha,beta (where m = 1,2,3). These results suggest the presence of minor sequence variants in acharan sulfate containing unsulfated iduronic acid having the structure -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp(1-->.

Animals↗

PubMed1998

Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication.

Individuals with the recessive condition trimethylaminuria exhibit variation in metabolic detoxication of xenobiotics by hepatic flavin-containing monooxygenases. We show here that mutations in the human flavin-containing monooxygenase isoform 3 gene ( FMO3 ) impair N -oxygenation of xenobiotics and are responsible for the trimethylaminuria phenotype. Three disease-causing mutations in nine Australian-born probands have been identified which share a particular polymorphic haplotype. Nonsense and missense mutations are associated with a severe phenotype and are also implicated in impaired metabolism of other nitrogen- and sulfur-containing substrates including biogenic amines, both clinically and when mutated proteins expressed from cDNA are studied in vitro . These findings illustrate the critical role played by human FMO3 in the metabolism of xenobiotic substrates and endogenous amines.

Adolescent↗