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Biomedical subjects

Y Lin

Publications and source records attributed to Y Lin.

At least 37 records · Page 2Linked to original sources
PubMed2001

Benzyloxybenzaldehyde analogues as novel adenylyl cyclase activators.

Several benzyloxybenzaldehyde analogues were prepared and found to have significant inhibitory activity toward neutrophil superoxide formation. Consequently, these compounds were evaluated for cAMP-elevating capability. Among them, benzyloxybenzaldehyde (7), exhibiting activity equivalent to forskolin, was determined as an adenylyl cyclase activator since it elevates cAMP levels by activation of adenylyl cyclase but not by inhibition of phosphodiesterase. Having a chemical structure very different from known adenylyl cyclase activators, compound 7 is recommended by us for use as a new lead compound in the future development of adenylyl cyclase activators.

Adenylyl Cyclases↗
PubMed2001

Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system.

We have developed a novel technique for high-throughput simultaneous screening of multiple cytokine expression based on a protein array system. Our method has the advantage of showing the specificity of enzyme-linked immunosorbent assays, sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, the cytokine array membranes were created by spotting capture antibodies onto the membranes. The membranes were then incubated with biological samples such as conditioned media and patient's sera. The bound proteins were then recognized by biotin-conjugated antibodies and detected by horseradish peroxidase-conjugated streptavidin coupled with ECL. Experiments demonstrated that 24 cytokines from conditioned media and patient's sera could be simultaneously detected using this new approach. This methodology should allow us to develop many high-density protein array systems to detect a variety of proteins. To validate and quantitate the expression of key molecules in a wide range of samples, we have developed conditioned medium arrays to evaluate hundreds and even thousands of samples from individual cells and patients in a single microarray. The combinations of protein arrays and conditioned medium arrays or serum arrays will provide a powerful tool to identify the protein expression profiles and rapidly validate their expression in many types and numbers of samples.

Antibodies↗
PubMed2001

Inhibitory effects of orally administered green tea, black tea, and caffeine on skin carcinogenesis in mice previously treated with ultraviolet B light (high-risk mice): relationship to decreased tissue fat.

Treatment of SKH-1 hairless mice with ultraviolet B light (UVB; 30 mJ/cm(2)) twice a week for 22 weeks resulted in tumor-free animals with a high risk of developing malignant and nonmalignant skin tumors during the next several months in the absence of additional UVB treatment (high-risk mice). Oral administration of green tea or black tea (6 mg tea solids/ml) to UVB-pretreated high-risk SKH-1 mice for 23 weeks after stopping UVB treatment decreased the number of tumors/mouse, decreased the size of the parametrial fat pads, and decreased the thickness of the dermal fat layer away from tumors and directly under tumors. Administration of the decaffeinated teas had little or no effect on these parameters, and adding caffeine (equivalent to the amount in the regular teas) to the decaffeinated teas restored their inhibitory effects. Administration of caffeine alone also decreased the number of tumors/mouse, the size of the parametrial fat pads, and the thickness of the dermal fat layer away from tumors and under tumors. Using data from individual mice and linear regression and correlation analysis, we found a highly significant positive correlation between the thickness of the dermal fat layer away from tumors and the number of tumors/mouse (r = 0.34; P = 0.0001), but the correlation between average tumor size/mouse and the thickness of the dermal fat layer away from tumors was weak (r = 0.16; P = 0.034). The results suggested that p.o. administered tea or caffeine may have decreased tumor multiplicity in part by decreasing fat levels in the dermis. Additional analysis revealed that oral administration of caffeinated beverages (green tea, black tea, decaffeinated green tea plus caffeine, decaffeinated black tea plus caffeine, or caffeine alone) decreased the thickness of the dermal fat layer under large tumors to a much greater extent than under small tumors. This is the first demonstration of a close association between inhibition of carcinogenesis and the lowering of tissue fat levels by a chemopreventive agent.

Adipose Tissue↗
PubMed2001

Pharmacological blockade of brain alpha1-adrenoceptors as measured by ex vivo [3H]prazosin binding is correlated with behavioral immobility.

The present studies examined the relationship between the blockade of central alpha1-adrenoceptors, as measured by ex vivo binding of [3H]prazosin in the cerebral cortex and the inhibition of behavioral activation to a mildly novel environment (cage change). It was found that intraventricular (i.v.t.) terazosin, a saline-soluble alpha1-adrenoceptor antagonist, dose dependently inhibited both ex vivo cortical binding and behavioral activation and that there was a highly significant positive correlation between the two with a slope near unity. Prazosin, a nonsaline soluble antagonist which had to be given intraperitoneally (i.p.), was much less potent at blocking both behavioral activity and cortical ex vivo binding, although it blocked ex vivo binding in the lung, indicating that it was effective peripherally but did not readily enter the brain. Despite this, however, the inhibition of cortical binding and behavioral activation that i.p. prazosin did produce were highly correlated with each other and had a slope near unity as with terazosin, whereas the more potent inhibition of lung binding was less well correlated with behavioral inhibition and had a slope significantly less than one. These results confirm our earlier studies, which have shown that alpha1-adrenoceptor activity is essential for gross and fine motor behavior in the mouse and that prazosin, which is used extensively in behavioral research, has difficulty entering the mouse brain.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2001

Disruption of the EF-2 kinase/Hsp90 protein complex: a possible mechanism to inhibit glioblastoma by geldanamycin.

Glioblastoma multiforme is the most treatment-resistant brain tumor. Elongation factor-2 (EF-2) kinase (calmodulin kinase III) is a unique protein kinase that is overexpressed in glioma cell lines and in human surgical specimens. Several mitogens activate this kinase and inhibitors block mitogen activation and produce cell death. Geldanamycin (GA) is a benzoquinone ansamycin antibiotic that disrupts Hsp90-protein interactions. Because EF-2 kinase is chaperoned by Hsp90, we investigated the effects of GA on the viability of glioma cells, the expression of EF-2 kinase protein, and the interaction between Hsp90 and EF-2 kinase. GA was a potent inhibitor of the clonogenicity of four glioma cells lines with IC(50)s ranging from 1 to 3 nM. 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic and less potent derivative of GA, inhibited the clonogenicity of glioma cells with IC(50) values of 13 nM in C6 cells and 35 nM in T98G cells. Treatment of cell lines for 24-48 h of GA or 17-AAG disrupted EF-2-kinase/Hsp90 interactions as measured by coimmunoprecipitation, resulting in a decreased amount of recoverable kinase in cell lysates. The ability of GA to inhibit the growth of glioma cells was abrogated by overexpressing EF-2 kinase. In addition, 17-AAG significantly inhibited the growth of a glioma xenograft in nude mice. These studies demonstrate for the first time the activity of GAs against human gliomas in vitro and in vivo and suggest that destruction of EF-2 kinase may be an important cytotoxic mechanism of this unique class of drug.

Animals↗
PubMed2001

Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44.

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E(2), and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.

Adenocarcinoma↗
PubMed2001

Generation of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometric sensitivity.

Arrays of microelectrospray emitters were fabricated on polycarbonate substrates using a laser etching technique. Stable multielectrosprays were successfully generated in the liquid flow rate range relevant to mass spectrometric applications. Comparison of electrosprays generated from the microfabricated emitter array and conventional fused-silica capillaries showed similar spray characteristics and reliability. Higher total electrospray ion currents were observed as the number of electrosprays increased at a given total liquid flow rate. Consistent with the theoretical prediction, the total spray current at a constant total liquid flow rate was shown experimentally to be approximately proportional to the square root of the number of electrosprays. It is further projected that when total flow rate is optimized the maximum achievable total current will be proportional to the number of emitters. Evaluation of the multielectrospray device using a triple quadrupole mass spectrometer showed a factor of 2-3 sensitivity enhancement for the spray numbers ranging from two to nine compared to a conventional single electrospray ionization source under the same operating conditions.

Algorithms↗
PubMed2001

Structural basis for the functional switch of the E. coli Ada protein.

The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA. This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.

Bacterial Proteins↗
PubMed2001

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.

Amino Acid Sequence↗
PubMed2001

Structure and functionality of a designed p53 dimer.

P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.

Binding Sites↗
PubMed2001

Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants.

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.

Acute Disease↗
PubMed2001

[Application of distraction osteogenesis in correction of hemifacial microsomia].

OBJECTIVE: To study the effect of distraction osteogenesis of mandibular ramus in correcting hemifacial microsomia. METHODS: From December 1997 to March 2000 two kinds of mandibular distractors made in Germany and China were used in 11 cases with hemifacial microsomia aged 4.5 approximately 23 years (on average 14.1 years). Orthodontic treatment was given during the distraction. LeFort I osteotomy and genioplasty were performed in 5 cases and 4 cases respectively. RESULTS: The treated mandibular rami were lengthened 15 approximately 30 mm. No infection and dysosteogenesis was found. In only one case, the distractor failed to function at the third day of treatment and was replaced. The distractor was removed after 3 months while new bone was formed in all cases. All of the patients and their family members expressed their satisfaction with the results of treatment. CONCLUSION: Mandibular distraction osteogenesis is the most effective procedure for correcting hemifacial microsomia.

Adolescent↗
PubMed2001

Neuroprotection from glutamate toxicity with ultra-low dose glutamate.

The protective effects of ultra-low doses (ULD) of glutamate against glutamate toxicity was studied in primary rat spinal, cortical and cerebellar neurons. Neurons were exposed to four subtoxic, ultra-low concentrations of glutamate (10(-18) M, 10(-20)M, 10(-22) M and 10(-30) M) for 72 h and then subsequently challenged with toxic concentrations (25 microM) of glutamate. Neuron viability was consistently 10% higher in spinal and cortical neurons pre-exposed to glutamate concentrations of 10(-18) M and 10(-22) M, and in cerebellar neurons pre-exposed to 10(-20) M and 10(-30) M. Using laser scanning confocal microscopy and the fluorescent calcium probe fluo-3, we found no alterations in intracellular calcium dynamics in the protected cells. This protective effect is consistent with a growing body of evidence for tolerance induced by low-dose toxin exposure but is the first time that such tolerance has been demonstrated with ultra-low glutamate exposure. Our data show that pre-exposure of neuronal cells to ULD glutamate can protect against subsequent exposure to toxic levels of glutamate.

Animals↗
PubMed2001

Chromosomal localization, expression pattern, and promoter analysis of the mouse gene encoding adipocyte-specific secretory protein Acrp30.

Acrp30 is an abundantly expressed secretory protein exclusively synthesized in adipose tissue. Due to the dysregulation in various forms of obesity in humans and mice and its strong structural similarity to TNFalpha, it is currently under study as an important molecule involved in whole body energy homeostasis. Here we describe the sequence of mouse Acrp30 locus, define the intron/exon boundaries and map the gene to the telomere of mouse chromosome 16, syntenic to the human chromosomal locus 3q27. We demonstrate that alternative polyadenylation gives rise to two distinct mRNA species. We also show that Acrp30 expression is induced only at the late stages of mouse embryonic development. Finally, we have characterized the mouse Acrp30 promoter in tissue culture cells. We propose that Acrp30 promoter has the potential to drive strong adipocyte specific heterologous gene expression in transgenic mice.

3T3 Cells↗
PubMed2001

Stabilization and activation of p53 by the coactivator protein TAFII31.

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.

3T3 Cells↗
PubMed2001

Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II.

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Animals↗
PubMed2001

Bax accelerates tumorigenesis in p53-deficient mice.

Bax is a Bcl-2 family member that promotes apoptosis and counters the protective effect of Bcl-2. Bax is a downstream effector of p53-induced apoptosis and is transcriptionally regulated by p53. Moreover, the introduction of Bax deficiency accelerates the onset of tumors in transgenic mice expressing truncated large T antigen. These results implicate Bax as a tumor suppressor. Consequently, we asked whether the levels of Bax expression would influence tumor development by comparing Bax-deficient and Bax transgenic mice in the presence or absence of p53. We found that Bax-deficient mice did not display an increased incidence of spontaneous cancers when followed for > 1.5 years. In addition, Bax-deficiency did not further accelerate oncogenesis in mice also deficient in p53. We generated Lck(pr)-Bax transgenic mice to examine the effects of overexpressed BAX on T-cell development and tumorigenesis. Lck(pr)-Bax mice show increased apoptosis consistent with the pro-apoptotic function of Bax. The introduction of p53-deficiency did not interfere with BAX-induced apoptosis; this is consistent with BAX operating downstream or independent of p53. However, we found that Lck(pr)-Bax/p53-deficient mice have an increased incidence of T-cell lymphomas when compared with p53-deficient mice. The Lck(pr)-Bax transgenic mice have an increased percentage of cells in cycle. These findings extend previous work suggesting that Bcl-2 family proteins regulate proliferation as well as cell death. We conclude that BAX-induced proliferation is synergistic with a defect in apoptosis contributed by p53-deficiency. Thus, the dual roles of BAX can either accelerate or inhibit tumorigenesis depending on the genetic context.

Animals↗