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Biomedical subjects

Y Lin

Publications and source records attributed to Y Lin.

At least 379 records · Page 21Linked to original sources
PubMed1997

The transactivation and p53-interacting functions of hepatitis B virus X protein are mutually interfering but distinct.

Transactivation of viral and host genes expression by hepatitis B virus X protein (HBx) is believed to be involved in hepatocarcinogenesis. The interaction of HBx with the tumor suppressor p53 and its inhibitory effect on p53 functions have been reported recently. However, the question of whether p53 is directly involved in HBx transactivation has not yet been addressed. In this study, we delineated the interaction sites of HBx and p53 using far-Western blotting and glutathione S-transferase-resin pull-down assays. The results indicate that the HBx-binding sites are located within the oligomerization and specific DNA-binding domains of p53 and that the p53-binding site was confined to a small region in the HBx transactivation domain. Mutual interference of the transactivations by HBx and p53 was detected by CAT assays in a transient transfection system. Strikingly, transactivation by HBx was observed in the p53-negative cells, Saos-2 and Hep3B, indicating that the transactivation and the p53-inhibiting functions of HBx are mutually interfering but distinct.

Binding Sites↗
PubMed1997

Prolonged pre-exposure to 1-aminocyclopropanecarboxylic acid protects against subsequent glutamate toxicity in vitro.

Sustained 20 h pre-exposure to 1 mM 1-aminocyclopropanecarboxylic acid (ACPC, which was removed 30 min before addition of 25 microM glutamate) significantly reduced the subsequent neurotoxicity of glutamate in cultured forebrain and cerebellar neurons. The magnitude of neuronal protection was further enhanced if the neurons pretreated with ACPC were re-exposed to ACPC during glutamate challenge. These results closely resemble earlier findings with cultured spinal cord neurons and indicate that these primary cell culture preparations might be suitable for the assessment of the mechanism(s) underlying chronic ACPC-induced modification of the NMDA receptor complex.

Amino Acids↗
PubMed1997

Identification of quantitative trait loci controlling levels of radiation-induced thymocyte apoptosis in mice.

Thymocyte apoptosis levels are higher in C57BL/6J mice than in C3Hf/Kam mice. Low-dose irradiation increases the numbers of thymocytes undergoing apoptosis, but the strain difference persists. We mapped three loci controlling radiation-induced thymocyte apoptosis levels in F2 intercross progeny of these strains. The strongest association of a genomic region with an apoptosis level occurred in a region of chromosome 11 known to harbor a locus (or loci) important in the pathogenesis of several rodent models of autoimmune disease. Additional loci influencing radiation-induced thymocyte apoptosis were identified on chromosomes 9 and 16. The genetic polymorphisms underlying these loci may have an evolutionary role in fine-tuning the apoptotic response in T cells and may be important in the etiology of lymphoproliferative disorders and autoimmunity.

Animals↗
PubMed1997

Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer.

Single-stranded DNA aptamers that recognize DNA polymerase from Thermus acquaticus (Taq pol) with high affinity have been described recently. These aptamers have been shown to efficiently inhibit the polymerase activity of Taq pol and are useful in enhancing the amplification efficiency of low copy number targets by the polymerase chain reaction (PCR). Aptamers selected to bind to Taq pol fell into two different sequence families and inhibited several DNA polymerases isolated from the Thermus species, including that from Thermus thermophilus (Tth pol). Aptamers from one sequence family inhibited the Stoffel fragment of Taq pol efficiently, whereas those from the other family did not. Truncated aptamers derived from two parent ligands from both families were combined to form a heterodimeric aptamer that effectively inhibited all three polymerases and were shown to be useful in detecting a low copy number target by PCR amplification. These data demonstrate that the combination of aptamers with different properties into a single molecule broadens their spectrum of utility.

Base Sequence↗
PubMed1997

High molecular weight kininogen peptides inhibit the formation of kallikrein on endothelial cell surfaces and subsequent urokinase-dependent plasmin formation.

A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 micromol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 micromol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 micromol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/ L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Amino Acid Sequence↗
PubMed1997

Factors involved in rejection of concordant xenografts in complement-deficient rats.

BACKGROUND: Factors that contribute to xenograft (Xg) rejection were investigated in complement C6-deficient (C-) PVG rats. METHODS: First and second hamster hearts were transplanted in C6-deficient and C6-sufficient PVG rats. Xenoantibody (XAb) formation, hemolytic C (CH50) activity and immunohistochemistry were studied. RESULTS: PVG C6-deficient rats rejected Xgs 3 days later than PVG C6-sufficient rats. Surprisingly, C activation participated in the rejection in PVG C- rats, as shown by partially recovered serum CH50 levels and deposition of C factors in the Xgs. As we found that cultured endothelial cells produced C6 in vitro, we hypothesized that Xg endothelial cells corrected the C6 defect in PVG C- rats. This was probably induced by IgM XAbs as: (1) it did not occur in immunosuppressed PVG C- rats in which XAb formation was prevented, and (2) transfer of IgM XAbs to naive, xenotransplanted PVG C- rats accelerated the recovery of CH50 and concomitantly Xg rejection. Thirty days after rejection of a first Xg, when no IgM XAbs or CH50 activity but high levels of IgG XAbs were detected in PVG C- rats, second Xgs underwent a hyperacute rejection. This time, complement was not involved, as no serum CH50 nor C deposition was found in the Xg. Instead, IgG antibody-dependent cellular cytotoxicity was involved as: (1) IgG XAbs were deposited in the Xg and (2) hyperacute rejection was induced in naive PVG C- rats by transfer of IgG XAbs, and (3) this rejection was delayed to 5+/-3 days if the adoptive hosts were first irradiated. CONCLUSIONS: In the face of a defect of host C factors, IgM XAb may induce cells of the Xg to secrete C factors which may correct the C defect of the host. Even if activation of lytic C can be prevented, IgG XAb may still provoke an acute Xg rejection by antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1997

Use of the methylxanthine derivative A802715 in transplantation immunology: II. In vivo experiments.

BACKGROUND: We have previously demonstrated in vitro that the methylxanthine derivative A802715 suppresses the cyclosporine (CsA)-resistant "signal two"-dependent pathway of T cell activation and hence acts synergistically with CsA. Here, this synergism was further investigated in vivo in rats. METHODS: Primary cardiac allografts were placed in the neck, and secondary grafts were transplanted intra-abdominally. A802715 was given orally for 30 days or by continuous intravenous infusion via a mini-osmotic pump for 2 weeks. CsA was given orally for up to 30 days. T cell responses were examined in vitro using mixed lymphocyte reaction, concanavalin A whole blood, and cell-mediated lympholysis assays. RESULTS: In a major histocompatibility complex incompatible WKAH-->PVG combination, neither oral CsA (7.5 mg/kg/day) nor oral A802715 (100 mg/kg/day) was able to prolong graft survival. However, a combination of both drugs, given at the same dose, sustained graft survival during treatment. A similar synergism was not obtained with pentoxifylline, another methylxanthine derivative. The synergism between A802715 and CsA could be further increased by using a continuous intravenous infusion of A802715, since (1) lower doses of A802715 (20 mg/kg/day) and CsA (5 mg/kg/day) could be used, and (2) six of seven grafts survived permanently. In a major histocompatibility complex compatible Wag/Rij-->R/A combination, similar synergistic effects and permanent graft survival could also be obtained by oral A802715 (100 mg/kg/day) in combination with a low dose of CsA (2.5 mg/kg/day). In both strain combinations, long-term survivors accepted donor-type but rejected third-party second grafts in the absence of immunosuppression. This specific tolerance was not related to clonal deletion nor anergy, as recipient lymphocytes proliferated normally in the anti-donor mixed lymphocyte reaction. Instead, a defect in generating specific cytotoxic T lymphocytes was involved. CONCLUSIONS: A802715 synergizes with CsA in vivo to induce specific transplantation tolerance and hence should be considered as a promising new immunosuppressant.

Administration, Oral↗
PubMed1997

Use of the methylxanthine derivative A802715 in transplantation immunology: I. Strong in vitro inhibitory effects on CD28-costimulated T cell activities.

BACKGROUND: Recently, methylxanthines such as pentoxifylline (PTX) were shown to be immunosuppressive in vitro. Unfortunately, when used in transplant patients, PTX was poorly active as an immunosuppressant. Here we report that the new methylxanthine derivative A802715 not only is more active than PTX, it also suppresses the cyclosporine (CsA)-resistant "signal two"-dependent pathway of T cell proliferation, making it an interesting drug to associate with CsA. METHODS: "Signal one"- and "signal two"-dependent T cell activation was investigated with purified human T cells stimulated with immobilized anti-CD3 or anti-CD28 monoclonal antibody (mAb) plus phorbol myristate acetate (PMA) or with a 3T6 mouse fibroblast cell line presenting anti-CD3 mAb on transfected human Fcgamma receptors II (FcgammaRII) in the presence or absence of transfected B7-1 (CD80) molecules. RESULTS: A802715 was more immunosuppressive in the mixed lymphocyte reaction (MLR) than PTX. A802715 dose-dependently suppressed polyclonal signal one-dependent T cell activation induced by anti-CD3 mAb/PMA. In addition, A802715 also suppressed signal two-dependent T cell proliferation induced by anti-CD28 mAb/PMA. The expression of the interleukin-2 receptor on T cells stimulated by anti-CD3 mAb presented on 3T6/FcgammaRII cells was equally well suppressed by A802715 and PTX. In contrast, interleukin-2 receptor or CD40L (gp39) expression by T cells after stimulation with the same anti-CD3 mAb- 3T6/FcgammaRII cells, but coexpressing transfected B7-1, was only suppressed by A802715. The anticipated synergism between A802715 and CsA was confirmed in MLR assays. Moreover, generation of cytotoxic T lymphocytes during MLR with Epstein-Barr virus-transformed B cells, which strongly express B7-1 and B7-2, was also inhibited by A802715. CONCLUSIONS: These in vitro data indicate that the A802715 (1) is a stronger immunosuppressant for T cells than PTX, (2) suppresses T cell activation pathways that are resistant to PTX or CsA, and (3) acts synergistically with CsA.

Animals↗
PubMed1997

Natural killer cell- and macrophage-mediated rejection of concordant xenografts in the absence of T and B cell responses.

Hyperacute, complement-mediated xenograft (Xg) rejection is a first major hurdle for xenotransplantation. Thereafter, cellular immunity, including T and B lymphocytes, NK cells, and macrophages may become involved as well. In the present study, hamster heart Xgs were performed in Leflunomide (LF)-treated nude rats. These animals, which are genetically T cell deficient, are known to have a strong NK activity. Moreover, xenoantibody (XAb) formation by B lymphocytes was previously shown to be blocked in nude rats by LF. Hence, this model was well suited to study the role of NK cells and macrophages in Xg rejection. Despite a total suppression of XAb formation, LF-treated nude rats rejected hamster heart Xgs at a same speed (3 days) as untreated nude rats. NK cells played a major role in this rejection process. Indeed, an NK cell-dominated cellular infiltration was noticed in the rejected Xgs and the addition of an anti-NK cell serum, anti-Asialo GM-1 (ASGM-1), to the LF treatment resulted in a significant delay of Xg rejection (from 3 days to 6 days; p < 0.0001) that seemed to be mediated by activated macrophages because 1) spleens from rejecting animals showed an increased percentage of macrophages (ED1+ or ED2+) and 2) rejected Xgs were infiltrated predominantly by macrophages. Moreover, splenocytes taken from rats that rejected Xgs despite a treatment with LF + ASGM-1 provoked an acceleration of Xg rejection when transferred to newly transplanted LF + ASGM-1-treated nude rats, and finally, the latter accelerating effect disappeared when macrophages were first depleted from the adoptively transferred splenocytes. This study thus shows that NK cells and macrophages can be activated in the absence of T cells or XAbs to directly reject Xgs.

Adoptive Transfer↗
PubMed1997

Neuroprotective sigma ligands attenuate NMDA and trans-ACPD-induced calcium signaling in rat primary neurons.

The effect of neuroprotective sigma ligands possessing a range of relative selectivity for sigma and phencyclidine (PCP) binding sites on N-methyl-D-aspartate (NMDA) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated calcium flux was studied in 12-15-day-old primary cultures of rat cortical neurons. In approximately 80% of the neurons tested, NMDA (80 microM) caused a sustained increase in intracellular calcium ([Ca2+]i). With the exception of R-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP) (previously shown not to be neuroprotective) all of the sigma ligands studied significantly altered NMDA-induced calcium dynamics. The primary effect of dextromethorphan, (+)-pentazocine, (+)-cyclazocine, (+)-SKF10047, carbetapentane, 1,3-di(2-tolyl) guanidine (DTG), and haloperidol was to shift the NMDA response from a sustained, to either a biphasic or a transient, calcium event. In contrast to NMDA, the primary response observed in 62% of the neurons treated with trans-ACPD (100 microM) was a transient elevation in [Ca2+]i. Here, however, only the highly selective neuroprotective sigma ligands (i.e., those lacking substantial PCP binding affinity) significantly decreased the number of transient responses elicited by trans-ACPD whereas the PCP-related sigma ligands such as dextromethorphan, (+)-SKF10047 and (+)-cyclazocine were ineffective. Unexpectedly, (+)-3-PPP potentiated trans-ACPD activity. These results demonstrating attenuating effects of sigma ligands on NMDA-stimulated neuronal calcium responses agree with earlier studies using glutamate and KCl and identify a sigma receptor modulation of functional NMDA responsiveness. Furthermore, the ability of sigma ligands to attenuate NMDA-, trans-ACPD- and KCl-evoked neuronal calcium dynamics indicates that the receptor mechanisms mediating sigma neuroprotection comprise complex interactions involving ionotropic, metabotropic, and even voltage-gated calcium signaling processes.

Animals↗
PubMed1997

Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation.

Transcription of c-myc in plasma cells, which are terminally differentiated B cells, is repressed by plasmacytoma repressor factor. This factor was identified as Blimp-1, known for its ability to induce B cell differentiation. Blimp-1 repressed c-myc promoter activity in a binding site-dependent manner. Treatment of BCL1 lymphoma cells with interleukin-2 (IL-2) plus IL-5 induced Blimp-1 and caused a subsequent decline in c-Myc protein. Ectopic expression of Blimp-1 in Abelson-transformed precursor B cells repressed endogenous c-Myc and caused apoptosis; Blimp-1-induced death was partially overcome by ectopic expression of c-Myc. Thus, repression of c-myc is a component of the Blimp-1 program of terminal B cell differentiation.

Animals↗
PubMed1997

Hepatitis B virus X protein is a transcriptional modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5.

Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferase-resin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.

Gene Expression Regulation, Viral↗
PubMed1997

Localization of structural elements of bee venom phospholipase A2 involved in N-type receptor binding and neurotoxicity.

We have shown previously that neurotoxic venom secretory phospholipases A2 (sPLA2s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins. The sPLA2 found in honey bee venom is neurotoxic and binds to this receptor with high affinity. In this paper, we have used a number of mutants of bee venom sPLA2 produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors. Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA2 structure do not significantly modify the binding properties. Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity. These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA2s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA2 as the N-type receptor recognition domain.

Animals↗
PubMed1997

TNF translationally modulates the expression of G1 protein alpha(i2) subunits in human polymorphonuclear leukocytes.

Priming of polymorphonuclear leukocyte responses to chemoattractants by TNF plays an important role in host defenses and inflammatory responses. TNF-induced priming is associated with an 80% increase in the membrane density of G alpha(i2) protein that is coupled to chemoattractant receptors. The present study examines the hypothesis that TNF stimulates increased synthesis of alpha(i2). Within 10 min of addition, TNF stimulated a significant increase in total cellular G alpha(i2), as determined by pertussis toxin-catalyzed ADP ribosylation, which was blocked by the translation inhibitor cycloheximide. Immunoprecipitation of biosynthetically labeled alpha(i2) showed that TNF increased alpha(i2) synthesis by about 20% at 10 min. Nuclear run-ons showed no change in alpha(i2) mRNA synthesis in TNF-treated cells; however, steady state alpha(i2) mRNA levels were reduced following a 10-min exposure to TNF. Pretreatment with cycloheximide prevented the TNF-induced reduction in steady state alpha(i2) mRNA levels. Therefore, TNF stimulates alpha(i2) protein synthesis and mRNA degradation in the same time frame as priming. The increased alpha(i2) synthesis results from increased translation, not transcription, of alpha(i2) mRNA. Simultaneous G alpha(i2) protein synthesis and mRNA degradation provide a mechanism by which TNF priming is associated with a rapid, self-limiting increase in G protein expression.

Biological Transport↗
PubMed1997

In vivo electron spin resonance analysis of nitroxide radicals injected into a rat by a flexible surface-coil-type resonator as an endoscope- or a stethoscope-like device.

The flexible surface-coil-type resonator (FSCR) operating in a 700 MHz microwave electron spin resonance (ESR) system was applied to measure the nitroxide radicals at a specific area in rats. The FSCR was composed of a single-turn coil with a diameter of 5 mm and two flexible coaxial lines of 450 mm in length. For an endoscope-like application, the FSCR was inserted into the rectum of the rat and sequential changes in the ESR signals of the intravenously injected nitroxide radical (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl; TEMPOL or 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy; carbamoyl-PROXYL) were measured. The ESR signal intensity of the nitroxide decreased according to first-order kinetics. For a stethoscope-like application, the FSCR was placed at several sites on the abdominal skin of the rats receiving a subcutaneous injection of carbamoyl-PROXYL, and diffusion and/or metabolism of the radical in the skin was observed.

Animals↗
PubMed1997

Load assessment of the static posture and weight holding in terms of pulmonary ventilation and heart rate.

The aim of this study was to explore appropriate index for load evaluation by analyzing the changes of pulmonary ventilation (VE) and heart rate (HR) in 35 student volunteers under the condition of static posture and weight holding. Each subject was asked to perform 7 kinds of static postures and 2 kinds of weight holdings. The HR was continuously measured by a remote detecting device. VE was recorded by a ventimeter for an average of 5 min. The results showed that HR was positively correlated to VE under static activities (r = 0.89), which suggested that VE as well as HR could to some degrees reflect the static load. It was better to use VE to indicate the load of weight holdings than to use HR, which, on the other hand, was more sensible in reflection of the posture load. It is better to use relative value of both of VE and HR to reflect the static load. In addition, arm-overheaded posture increase HR more significantly than any other activities.

Adult↗
PubMed1997

Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.

Adenosine↗