Biomedical subjects

Y Lin

Publications and source records attributed to Y Lin.

At least 55 records · Page 3Linked to original sources

PubMed2001

Mutational analysis of the structure and functions of hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication. To further understand the structure and functions of NS5B, we introduced a series of 27 clustered and 19 point substitution mutations within and outside the well-known motifs conserved among RdRP by alanine scanning and investigated effects of these mutants on enzymatic activity of NS5B. Surprisingly, most of the mutations (22 of 27 clustered mutants) do not affect RdRP activity at all, indicating that the side chains of the corresponding amino acid residues are dispensable for the catalytic activity. On the other hand, 4 mutants, cm20t, cm194t, cm2t, and cm3t, are defective in RdRP activity. By further analysis with point mutations within these regions, E18, Y191, C274, Y276, and H502 were determined to be critical for the RdRP activity. Y276 was also shown to be critical for RNA template/primer association, although 3 amino acid sequences were identified to be important for RNA template binding by RNA-filter binding assays. Finally, 4 discontinuous sequences of NS5B (aa139-145, aa149-155, aa 365-371, and aa 382-388) were found to be essential for binding to NS5A as determined by glutathione S-transferase (GST)-pull down assays using GST-NS5A and FLAG-NS5B expressed in cotransfected cells, and GST-pull down assay in vitro. In light of the crystal structure models of NS5B recently reported, our results indicate that the RdRP activity of NS5B requires the longer loop and the helix located at the distal of the thumb, which are unique among RdRPs as well as reverse transcriptases.

Amino Acid Sequence↗

PubMed2001

Preclinical evaluation of a humanized NR-LU-10 antibody-streptavidin fusion protein for pretargeted cancer therapy.

A humanized single chain Fv antibody fragment specific to the EGP40 antigen was genetically engineered as a streptavidin fusion (scFvSA) for use in pretargeted radioimmunotherapy. The scFvSA construct was expressed as a soluble, tetrameric species in the Escherichia coli periplasm at 110-140 mg/liter. The fusion protein was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of 50-60%. Characterization of the purified protein by SDS-PAGE, light scattering, and size exclusion chromatography demonstrated that the fusion protein was tetrameric with a molecular weight of approximately 172,000. Competitive immunoreactivity assays showed a two-fold greater binding to the antigen than the comparable whole antibody. The purified protein had a biotin disassociation rate identical to recombinant streptavidin and bound an average of three of four possible biotins per molecule. The radiolabeled fusion protein showed a faster blood clearance rate in normal mice than the corresponding whole antibody-streptavidin chemical conjugate. Tumor-specific targeting of a subsequently administered radionuclidechelate/biotin molecule was demonstrated in nude mice bearing SW1222 human colon carcinoma xenografts. A single dose of 800 microCi of 90Y-DOTA-biotin produced cures in mice with established subcutaneous human small cell lung or colon cancer xenografts.

Animals↗

PubMed2001

Capture of DNA sequences at double-strand breaks in mammalian chromosomes.

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.

Animals↗

PubMed2001

Women on the editorial boards of major journals.

PURPOSE: To determine the percentages of women on the editorial boards of general and specialty medical journals in comparison with the numbers of women physicians in the journals' respective specialties. METHOD: The numbers of women editors, deputy editors, assistant editors, and members of editorial boards of 12 major journals in 1999 were counted and compared with the percentages of women physicians in the journals' specialties, as published by the American Medical Association. RESULTS: Parity between the percentages of women on editorial boards of specialty journals and women physicians in the journals' specialties was found for five journals. Only one journal had more women on the editorial board than there were women physicians in the specialty. CONCLUSIONS: Fewer than half of the journals studied had parity between the percentages of women members of editorial boards and the percentages of women physicians in the specialties. Parity should be maintained to accurately reflect the numbers of women physicians in these fields.

American Medical Association↗

PubMed2001

Dynamic localization of rop GTPases to the tonoplast during vacuole development.

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.

Endoplasmic Reticulum↗

PubMed2001

The Rop GTPase switch controls multiple developmental processes in Arabidopsis.

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.

Arabidopsis↗

PubMed2001

Reduced antibody response to streptavidin through site-directed mutagenesis.

Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.

Amino Acid Sequence↗

PubMed2001

The alpha and beta subunits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF.

The activation of IkappaB kinase (IKK) is a key step in the nuclear translocation of the transcription factor NF-kappaB. IKK is a complex composed of three subunits: IKKalpha, IKKbeta, and IKKgamma (also called NEMO). In response to the proinflammatory cytokine tumor necrosis factor (TNF), IKK is activated after being recruited to the TNF receptor 1 (TNF-R1) complex via TNF receptor-associated factor 2 (TRAF2). We found that the IKKalpha and IKKbeta catalytic subunits are required for IKK-TRAF2 interaction. This interaction occurs through the leucine zipper motif common to IKKalpha, IKKbeta, and the RING finger domain of TRAF2, and either IKKalpha or IKKbeta alone is sufficient for the recruitment of IKK to TNF-R1. Importantly, IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1, as this occurs efficiently in IKKgamma-deficient cells. Using TRAF2(-/-) cells, we demonstrated that the TNF-induced interaction between IKKgamma and the death domain kinase RIP is TRAF2 dependent and that one possible function of this interaction is to stabilize the IKK complex when it interacts with TRAF2.

Animals↗

PubMed2001

Inhibition of insulin signaling and glycogen synthesis by phorbol dibutyrate in rat skeletal muscle.

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 microM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-alpha, -betaI, -theta, and -epsilon, and probably -betaII, from the cytosol to membranes. Preincubation with 1 microM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3'-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2 was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2 are unaffected.

Animals↗

PubMed2001

Requirement of protein tyrosine phosphatase SHP2 for NO-stimulated vascular smooth muscle cell motility.

We have previously reported that nitric oxide (NO) increases the motility of differentiated cultured primary aortic smooth muscle cells from adult rats. There is little information on the role of protein tyrosine phosphatases in vascular biology. One such phosphatase, Src homology 2 phosphatase 2 (SHP2), is essential for motility. We tested the hypothesis that NO increases SHP2 levels via a cGMP-mediated mechanism and that this effect is necessary for NO-stimulated cell motility. Here we report that two different NO donors increased SHP2 protein levels and enzyme activity. This effect was mimicked by several cGMP agonists and blocked by an inhibitor of guanylyl cyclase. Specific decrease of SHP2 protein levels via the use of antisense oligodeoxynucleotides (ODNs), but not several control ODNs attenuated the motogenic effect of NO, which indicates the involvement of SHP2 in NO-elicited motogenesis. S-nitroso-N-acetylpenicillamine failed to increase SHP2 protein levels in subcultured aortic smooth muscle cells. This provides a potential explanation for the lack of effect of NO on cell motility in dedifferentiated subcultured cells. These results support the hypothesis that NO-elicited upregulation of SHP2 via a cGMP-mediated pathway is necessary for NO-induced motogenesis in differentiated aortic smooth muscle cells.

Animals↗

PubMed2001

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C.

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.

Animals↗

PubMed2001

The influence of chronic antihypertensive treatment on the central pressor response in SHR.

UNLABELLED: We examined the influence of chronic antihypertensive treatment on the central pressor response in SHR. Adult male SHR were divided into 5 groups, i.e., those receiving 1) enalapril (Enal: 25 mg/kg/day in drinking water, n=12); 2) losartan (Los: 40 mg/kg/day, n=11); 3) candesartan (Cand: 4 mg/kg/day, n=12); 4) hydralazine+hydrochlorothiazide (H&H: 50+7.5 mg/kg/day, n=9); 5) vehicle ( CONTROL: n=9). At 4 weeks of treatment, hypertonic saline (0.25, 0.5 M) was intracerebroventricularly (i.c.v.) injected into conscious rats. Plasma catecholamines were measured before and after i.c.v. injection. On completion of the experiment, heart weight was measured, and angiotensin-converting enzyme (ACE) activity of the cerebrum was determined. All antihypertensive drugs elicited comparable reductions in systolic blood pressure, while heart rate was significantly higher in the H&H group than in the other groups during treatment. Pressor response to i.c.v. hypertonic saline (0.5 M) was significantly smaller in the Enal (12 +/- 3 mmHg) and Cand (11 +/- 2 mmHg) groups than in the Los (22 +/- 2 mmHg), H&H (16 +/- 2 mmHg), and CONTROL (29 +/- 5 mmHg) groups. Plasma catecholamines did not differ among the groups. Heart weight was lowest in the Enal group, followed by the Los and Cand groups. ACE activity of the cerebrum was significantly decreased in the Enal group. The results suggest that chronic treatment with various antihypertensive drugs differentially alters the central pressor response in SHR, and enalapril and candesartan are effective in attenuating this response.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

Algorithms↗

PubMed2001

Plasmablastic lymphoma of the lung: report of a unique case and review of the literature.

Non-Hodgkin lymphomas associated with acquired immunodeficiency syndrome are heterogeneous. Recently, a novel subtype of non-Hodgkin lymphoma occurring mostly in patients with acquired immunodeficiency syndrome has been described and designated as plasmablastic lymphoma. The histomorphologic and immunophenotypic findings of this distinct subtype of non-Hodgkin lymphoma have been characterized previously. Most patients present with oral cavity involvement. We report a case of plasmablastic lymphoma presenting as a lung tumor. To our knowledge, this is the first case report of this unusual subtype of diffuse large B-cell lymphoma in this location.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Substrate inhibition kinetics for cytochrome P450-catalyzed reactions.

Most cytochrome P450 (P450 or CYP)-catalyzed reactions are adequately described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), which are usually determined by a saturation profile of velocity of product formation versus substrate concentration. In turn, these parameters may be used to predict pharmacokinetics. However, some P450 enzymes exhibit atypical or non-Michaelis-Menten kinetics, due largely to substrate inhibition at higher concentrations of substrate. Although the mechanism of substrate inhibition is unknown, ignoring it and truncating the data can lead to erroneous estimates of kinetic parameters. In the present study, 13 P450 marker substrates were examined with 10 recombinant P450 proteins, and 6 were found, to varying degrees, to exhibit substrate inhibition. To understand the nature of the inhibition, a kinetic model was proposed (assuming that two binding sites exist on the enzyme) and used to fit the experimental data. The derived data indicated that 1) the K(I) values (substrate inhibition) were approximately 1.2- to 10-fold greater than the respective K(S) values; 2) both K(S) and K(I) values may be affected by the interaction of the two bound substrates within the enzyme, exhibited by a factor alpha (alpha = 5.1-23.3); and 3) enzyme activity was inhibited markedly (39-97%) at excess concentrations of the substrates (beta = 0.03-0.61). These findings suggest that substrates have access to both the inhibitory site and catalytic site simultaneously (K(I) > K(S)). Furthermore, the two sites, in the presence of substrate, can interact with each other. Therefore, the degree of inhibition of the enzyme is dependent on the concentration of the substrate (usually >K(I)) that sufficiently occupies the inhibitory site.

Antineoplastic Agents↗

PubMed2001

[Toxic effects of Cu, Zn and Mn on the inhibition of Chlorella pyrenoidosa's growth].

A standard method of algal bioassay for evaluating the toxicity of toxic chemicals was applied in the study. It was found that safety concentrations of Cu, Zn and Mn for Chlorella pyrenoidosa's growth were 31.8 micrograms/L, 65.0 micrograms/L and 5.5 mg/L, and 96 h-EC50 were 67.3 micrograms/L, 473.0 micrograms/L and 17.0 mg/L, respectively. According to both safety concentration and 96 h-EC50, the toxic sequences of these metals from high to low on the inhibition of C. pyrenoidosa's growth were Cu, Zn and Mn. The different affinities between different metal ions and algal cells are mainly responsible for the different inhibitions of C. pyrenoidosa's growth.

Chlorella↗

PubMed2001

[Treatment of dilute solution anilines-containing with displacement by extraction].

It was found that the biodegradability (indicated by BOD5/CODCr value of water solution containing anilire or chloroaniline declined with the increasing of their concentration. In order to bio-treat such water, decrease of the solute's concentration was necessary. Based on the research of organic solvents' and complexing agent's biodegradability, a series of extraction experiments were conducted for aniline and m-chloroaniline with complexing agent or physical solvent. The factors influencing the distribution ratio were discussed. The BOD5/CODCr value of raffinates showed that the raffinate can be treated by biological degradation without dilution if suitable solvent was choosed. It was proved that displacement by extraction had great potentiality for treating wastewater containing non-biodegradable organic contaminant.

Aniline Compounds↗

PubMed2001

Dendritic growth induced by BMP-7 requires Smad1 and proteasome activity.

Bone morphogenetic proteins (BMPs) induce dendritic growth in cultured sympathetic neurons; however, the signaling pathways that mediate this dendrite-promoting activity have not been previously characterized. Here we report studies of the signaling events that regulate the growth of these afferent processes. We find that Smad1 is expressed in sympathetic neurons and that BMPs rapidly induce its phosphorylation and translocation from the cytoplasm to the nucleus. Furthermore, a dominant negative form of Smad1 inhibits BMP-7-induced dendritic growth, suggesting a requirement for Smad1 activation in this biological activity of BMP-7. A physical interaction between Smad1 and components involved in the proteasome-mediated degradation system was detected with a yeast two-hybrid screen, thereby prompting an examination of the effects of proteasome inhibitors on dendritic growth. Lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth without adversely affecting either cell viability or axonal growth. Moreover, studies of transfected P19 cells suggest that the proteasome inhibitors directly block the effects of Smad1 on the transcriptional activity of the Tlx-2 promoter. These data indicate that BMP-induced dendritic growth requires Smad1 activation and involves proteasome-mediated degradation events.

Acetylcysteine↗