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Biomedical subjects

Y Lin

Publications and source records attributed to Y Lin.

At least 19 recordsLinked to original sources
PubMed1999

Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin.

We have investigated the molecular determinants responsible for alpha-bungarotoxin (alphaBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous alpha subunits, one highly sensitive to alphaBgtx block (alpha1) and the other, alphaBgtx-insensitive (alpha3). By replacing rat alpha3 residues 184-191 with the corresponding region from the Torpedo alpha1 subunit, we introduced a cluster of five alpha1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the alpha3 subunit. Functional activity and alphaBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric alpha3 subunit (alpha3/alpha1[5]) with either rat beta2 or beta4 subunits. Agonist-evoked responses of alpha3/alpha1[5]-containing receptors were blocked by alphaBgtx with nanomolar affinity (IC(50) values: 41 nM for alpha3/alpha1[5]beta2 and 19 nM for alpha3/alpha1[5]beta4). Furthermore, receptors containing the single point mutation alpha3K189Y acquire significant sensitivity to alphaBgtx block (IC(50) values: 186 nM for alpha3K189Ybeta2 and 179 nM for alpha3K189Ybeta4). Another alpha3 chimeric subunit, alpha3/alpha7[6], similar to alpha3/alpha1[5] but incorporating the corresponding residues from the alphaBgtx-sensitive alpha7 subunit, also conferred potent alphaBgtx sensitivity to chimeric receptors when co-expressed with the beta4 subunit (IC(50) value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the alphaBgtx-sensitive subunits alpha1 and alpha7 play a critical functional role in the interaction of alphaBgtx with nicotinic acetylcholine receptors sensitive to this toxin.

Amino Acid Sequence↗
PubMed1999

Accommodated xenografts survive in the presence of anti-donor antibodies and complement that precipitate rejection of naive xenografts.

Hamster hearts transplanted into transiently complement-depleted and continuously cyclosporin A (CyA)-immunosuppressed rats survive long-term despite deposition of anti-donor IgM Abs and complement on the graft vascular endothelium. This phenomenon is referred to as "accommodation." The hypothesis tested here is that accommodated xenografts are resistant to IgM Abs and complement that could result in rejection of naive xenografts. After first hamster hearts had been surviving in cobra venom factor (CVF) + CyA-treated rats for 10 days, a time when the anti-donor IgM Ab level was maximal and complement activity had returned to approximately 50% of pretreatment levels, naive hamster hearts or hamster hearts that had been accommodating in another rat for 14 days were transplanted into those rats carrying the surviving first graft. The naive hearts were all hyperacutely rejected. In contrast, a majority of regrafted accommodating hearts survived long-term. There was widespread Ab and activated complement deposition on the vascular endothelium of accommodating first hearts, second accommodating hearts, and rejected second naive hearts. However, only the rejected naive hearts showed extensive endothelial cell damage, myocardial necrosis, fibrin deposition, and other signs of inflammation. Accommodating first and second hearts but not rejected second naive hearts expressed high levels of the protective genes A20, heme oxygenase-1 (HO-1), bcl-2, and bcl-xL. These data demonstrate that accommodated xenografts become resistant to effects of anti-donor IgM Abs and complement that normally mediate rejection of xenografts. We hypothesize that this resistance involves expression by accommodated xenografts of protective genes.

Adoptive Transfer↗
PubMed1999

Camouflaging endothelial cells: does it prolong graft survival?

Camouflaging antigens on the surface of cells seems an appealing way to prevent activation of the immune system. We explored the possibility of preventing hyperacute rejection by chemically camouflaging endothelial cells (EC). In vitro as well as in vivo experiments were performed. First, the ability of mPEG coating to prevent antibody-antigen interactions was evaluated. Second, we tested the degree to which mPEG coating prevents activation of EC by stimuli such as TNF-alpha and LPS. Third, in vivo experiments were performed to test the ability of mPEG coating to prolong xenograft survival. We demonstrate that binding of several antibodies to EC or serum proteins can be inhibited by mPEG. Furthermore, binding of TNF-alpha as well as LPS to EC is blocked since mPEG treatment of EC inhibits the subsequent up-regulation of E-selectin by these stimuli. However, in vivo experiments revealed that currently this method alone is not sufficient to prevent hyperacute rejection.

Animals↗
PubMed1999

Identification of four novel ADAMs with potential roles in spermatogenesis and fertilization.

The ADAM (A Disintegrin And Metalloprotease) family is known to have important roles in various developmental systems, e.g., myogenesis and neurogenesis. In this study, we searched for ADAMs that may function in spermatogenesis or fertilization, and have cloned and sequenced four new mouse ADAM cDNAs: ADAM 24, ADAM 25, ADAM 26 and ADAM 27. The deduced amino acid sequences show that all four contain the complete domain organization common to ADAM family members. Messenger RNA for each of the four ADAMs was found only in the testis. The conserved zinc-dependent metalloprotease active site HEXGHXXGXXHD was found in the metalloprotease domain of three of the novel ADAMs, suggesting that they are testis-specific proteases, to which we give the alternative names: testase 1, ADAM 24; testase 2, ADAM 25; and testase 3, ADAM 26. Using RNA extracted from testes of pre-pubertal males of increasing age (8-40days), we found that adult levels of transcription, assessed in Northern blots, are reached by day 20 (ADAM 27), day 25 (ADAMs 24 and 25) and in the range day 25-50 (ADAM 26). These results suggest that each ADAM is transcribed in spermatogenic cells in a regulated pattern at a specific developmental stage.

ADAM Proteins↗
PubMed1999

Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A.

Gene transcription requires the release of inactive DNA from its packaging of histone proteins. Following the discovery of the first transcription-associated histone acetyltransferase, tetrahymena GCN5, it was shown that yeast GCN5 is recruited to the promoter and causes hyper-acetylation of histones and transcriptional activation of target genes, establishing a direct connection between histone acetylation and transcriptional activation. Many other important transcription regulators have been found to have histone acetyltransferase activity, including TAFII230/250, p300/CBP and its associated factor PCAF. Here we present the solution structure of the catalytic domain of tGCN5 (residues 47-210) in complex with coenzyme A. The structure contains two domains; the amino-terminal domain is similar to those of other GCN5-related N-acetyltransferases but the carboxy-terminal domain is not. Coenzyme A binds in a deep hydrophobic pocket between the two domains. Chemical shift changes upon titration with histone H3 peptides indicate a binding site at the domain boundary opposite to the coenzyme A site. The structural data indicate a single-step acetyl-transfer reaction mechanism catalysed by a hydrogen bond to the backbone amide group of leucine 126 and the side-chain carboxyl group of a conserved acidic residue.

Acetyltransferases↗
PubMed1999

Alternative splicing of a short cassette exon in alpha1B generates functionally distinct N-type calcium channels in central and peripheral neurons.

The N-type Ca channel alpha1B subunit is localized to synapses throughout the nervous system and couples excitation to release of neurotransmitters. In a previous study, two functionally distinct variants of the alpha1B subunit were identified, rnalpha1B-b and rnalpha1B-d, that differ at two loci;four amino acids [SerPheMetGly (SFMG)] in IIIS3-S4 and two amino acids [GluThr (ET)] in IVS3-S4. These variants are reciprocally expressed in rat brain and sympathetic ganglia (). We now show that the slower activation kinetics of rnalpha1B-b (DeltaSFMG/+ET) compared with rnalpha1B-d (+SFMG/DeltaET) channels are fully accounted for by the insertion of ET in IVS3-S4 and not by the lack of SFMG in IIIS3-S4. We also show that the inactivation kinetics of these two variants are indistinguishable. Through genomic analysis we identify a six-base cassette exon that encodes the ET site and with ribonuclease protection assays demonstrate that the expression of this mini-exon is essentially restricted to alpha1B RNAs of peripheral neurons. We also show evidence for regulated alternative splicing of a six-base exon encoding NP in the IVS3-S4 linker of the closely related alpha1A gene and establish that residues NP can functionally substitute for ET in domain IVS3-S4 of alpha1B. The selective expression of functionally distinct Ca channel splice variants of alpha1B and alpha1A subunits in different regions of the nervous system adds a new dimension of diversity to voltage-dependent Ca signaling in neurons that may be important for optimizing action potential-dependent transmitter release at different synapses.

Action Potentials↗
PubMed1999

An integrated microfabricated device for dual microdialysis and on-line ESI-ion trap mass spectrometry for analysis of complex biological samples.

A microfabricated dual-microdialysis device in a single integrated microfabricated platform was constructed using laser micromachining techniques for the rapid fractionation and cleanup of complex biological samples. On-line dual microdialysis and ESI-MS of biological samples was demonstrated using an ion trap mass spectrometer. The mass spectra obtained demonstrated the efficiency of dual microdialysis for removing both high-molecular-weight and low-molecular-weight species that interfere with effective ESI-MS analysis of target biopolymers. Signal-to-noise ratios were also greatly improved compared to direct sample infusion. In addition to its compactness, negligible dead volume, and robustness, the device can be used at a flow rate of only 200 nL/min, an order of magnitude lower than that obtained previously. This reduced sample consumption and improved sensitivity with ESI-MS. The results suggest the potential for integration of such microfabricated devices with other sample manipulations for the rapid ESI-MS analysis of complex biological samples.

Escherichia coli↗
PubMed1999

The Pex16p homolog SSE1 and storage organelle formation in Arabidopsis seeds.

Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Amino Acid Sequence↗
PubMed1999

Human hepatitis B virus X protein is detectable in nuclei of transfected cells, and is active for transactivation.

Subcellular localization and transactivation of human hepatitis B virus X protein (HBx), a plausible causative factor for hepatocellular carcinogenesis, were studied in transiently transfected cells. The transactivation was detected not only by the cis-element driven chloramphenicol acetyltransferase (CAT) assay but also by immunostaining of CAT protein cotransfected into human hepatoma cell line HepG2. Scanning fluorescence microscopy showed the majority of immunological signals of HBx to be at the perinuclear region of transfected cytoplasm. HBx was also clearly detectable in the nucleus, though less intensely expressed. This was confirmed by Western analysis and coimmunoprecipitation of HBx with transcription factor IIB (TFIIB) in subcellular fractionations. The percentage of HBx-positive cells coincided with that of CAT-positive cells, and confocal laser microscopy revealed the coexistence of CAT signals in GFP-HBx positive cells. The SV40 large T antigen nuclear localization signal (NLS) appended HBx, regardless of whether NLS was added to the N- or C-terminus, transactivated all the examined X-responsive elements (XRE) similarly as did wild-type HBx. Similar results were obtained in p53 negative Saos-2 cells. The detected nuclear HBx may be involved in modulating the transcription at the promoter level whereas the HBx in cytoplasm may be working through signal transduction pathways.

Cell Nucleus↗
PubMed1999

The gene for an abundant parasite coat protein predicts tandemly repetitive metal binding domains.

Immobilization antigens are highly abundant surface membrane proteins that coat the surface of hymenostomatid ciliates. While their function is unknown, recent studies with the common fish parasite, Ichthyophthirius multifiliis, suggest their involvement in a novel mechanism of humoral immunity involving an effect of antibody on parasite behavior. To gain further insight into the nature of these proteins, we have cloned a gene encoding the 48kDa i-antigen of I. multifiliis. Analysis of the gene (designated IAG48[G1]) reveals a single, uninterrupted reading frame that predicts a protein of 442 amino acids. Based on its deduced amino acid sequence, the protein contains hydrophobic amino acid domains at its N- and C-terminus that are characteristic of signal peptide and GPI-anchor addition sites, respectively. The most striking feature of the predicted protein, however, is a series of tandem repeats that spans most of its length. The repeats themselves are characterized by periodic cysteine residues that fall into register when the homologous segments are aligned. Interestingly, the spacing of cysteines (C-X2,3-C) within a framework of larger (C-X2-C-X20-C-X3-C-X20-C-X2-C) motifs is entirely consistent with the structure of known zinc-binding proteins. Finally, comparison of the coding sequence of the 48kDa i-antigen gene with a partial cDNA previously thought to encode this protein reveals nearly complete identity except at their 3' ends, suggesting that alternative forms of the antigen exist.

Amino Acid Sequence↗
PubMed1999

[Inhibition of Bcl-2 expression in HL60 cells by incubation with antisense phosphorothioate oligodeoxynucleotides].

OBJECTIVE: To observe whether the expression of Bcl-2 mRNA and protein in HL60 cells could be decreased by incubation with antisense phosphorothioate oligodeoxnucleotides (ASPO) at different concentrations (5micromol/L,10micromol/L,and 20micromol/L). METHODS: The expressions of Bcl-2 protein and mRNA in HL60 cells were individually determined by immunocytochemistry, flow cytometry and RT-PCR. RESULTS: In the presence of ASPO, the levels of Bcl-2 mRNA and protein could be decreased. The inhibitory effect appeared in the first 24 hours of incubation and became intense as the concentration increased and the time prolonged,but it abated after 72 hours of incubation. Inhibition was not noted in the presence of sense phosphorothioate oligodeoxynucleotides(SPO). CONCLUSION: The data suggested that ASPO could inhibit the expression of Bcl-2 mRNA and protein in HL60 cells and the inhibitory effect depended on the specific sequences, the concentration and the time of incubation, but it could not last very long.

HL-60 Cells↗
PubMed1999

Rejection of cardiac xenografts by CD4+ or CD8+ T cells.

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 +/- 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 +/- 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 +/- 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 +/- 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 +/- 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 +/- 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.

Adoptive Transfer↗
PubMed1999

Human hepatitis B virus X protein augments the DNA binding of nuclear factor for IL-6 through its basic-leucine zipper domain.

The X gene product of human hepatitis B virus, HBx, transactivates the expression of viral and cellular genes through a wide variety of cis elements, including the nuclear factor for IL-6 (NF-IL6) binding sites, although HBx does not appear to bind DNA directly. We previously reported that HBx transactivated the interleukin 8 promoter through NF-kappaB binding site and C/EBP-like binding site (NF-IL6 binding site). In this study, the interactions were examined between NF-IL6 and HBx using recombinant proteins. In a DNA-protein binding assay, the formation of a specific complex between NF-IL6 and a DNA probe harboring an NF-IL6 binding site was increased by the addition of either the full or the C-terminal 104 amino acids of HBx. A direct protein-protein binding assay (far-Western blot) revealed the direct interaction between the C-terminal 104 amino acids of HBx and the basic region-leucine zipper domain of NF-IL6. These results indicate that HBx alters the DNA-binding affinity of NF-IL6 through the direct interaction between the C-terminal domain of HBx and the basic region-leucine zipper domain of NF-IL6.

Binding Sites↗
PubMed1999

Mutation of structural determinants lining the N-methyl-D-aspartate receptor channel differentially affects phencyclidine block and spermine potentiation and block.

Spermine and other endogenous polyamines potentiate, block and permeate the N-methyl-D-aspartate receptor channel. To identify structural determinants of the N-methyl-D-aspartate channel that mediate spermine's actions, we generated mutant receptors with asparagine (N) to glutamine (Q) or arginine (R) substitutions in the selectivity filter of the channel. We demonstrate that mutation of the three critical asparagines in this domain differentially affects block by phencyclidine and both potentiation and block by spermine. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR1(011), N619 in NR1(100)) and N-to-Q mutations in the N and N + 1 sites (N595 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN receptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermine potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while the Q/ NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca2+ permeability and Mg2+ block of N-methyl-D-aspartate channels are also critical to block by spermine and phencyclidine. To examine the interaction of spermine and phencyclidine within the channel, we performed competition studies. Spermine appeared to compete with phencyclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na+ influx.

Algorithms↗
PubMed1999

Tissue folate binding protein levels in transgenic mice with tumors and in non-transgenic controls.

Localized folate deficiency may be a risk factor for cancer. Since, folate binding proteins (FBP) and reduced folate carrier proteins (RFC) mediate cellular transport of folate, we compared FBP concentrations in several organs from tumor-bearing transgenic (TBT) mice and tumor-free non-transgenic controls (NTC) of the same strain, age, and fed identical diets. Liver, spleen, brain, small intestine and kidney were individually homogenized in phosphate-buffered saline (PBS) and separated into membrane, cytoplasmic, mitochondrial/lysomal and nuclear fractions (confirmed with marker enzymes). Homogenates and fractions was analyzed for total protein, and FBP. We used rabbit anti-bovine milk antibody and ELISA to measure FBP. FBP concentrations in kidney, small intestine, and spleen of TBT mice were higher than those of NTC mice; the opposite was true in liver and lung. FBP seemed to be upregulated in kidneys (all fractions), small intestine (all fractions), and spleen (cytoplasmic and nuclear fractions only) of TBT mice compared to NTC mice; the opposite appeared true in liver (all fractions) and lung (all fractions). FBP concentrations in brain, heart, and muscle of TBT mice were not different from those in brain, heart and muscle of NTC mice. A longitudinal study will determine if these changes in FBP concentrations precede tumor onset.

Animals↗
PubMed1999

EPR imaging for in vivo analysis of the half-life of a nitroxide radical in the hippocampus and cerebral cortex of rats after epileptic seizures.

Recently, we developed an in vivo temporal electron paramagnetic resonance (EPR) imaging technique to be applied to the brain of a rat, into which a blood-brain barrier (BBB)-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally. This imaging technique made it possible to measure decay rates of a nitroxide radical in multiple regions of the brain simultaneously. Using this technique, the half-life of PCAM was estimated from the exponential decay of the signal intensity derived from the temporal EPR images in the hippocampus and cerebral cortex of rats after a kainic acid (KA)-induced seizure. The hippocampal half-life of PCAM after KA-induced seizures was significantly prolonged (p < .01), whereas the prolongation of the cortical half-life was not significant. These findings suggest that following a KA-seizure, the intrahippocampal ability to reduce the nitroxide radical is impaired, but the ability is intact in the cerebral cortex. This is the first in vivo quantitative EPR imaging study that has a bearing on the pathogenesis of KA-induced seizures in the brain.

Animals↗
PubMed1999

A new class of RAR subtype selective retinoids: correlation of pharmacological effects with receptor activity.

The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARbeta or RARbeta,gamma selectivity with weak RARalpha activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARgamma transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARbeta selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis.

Animals↗