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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 487 records · Page 27Linked to original sources
PubMed1999

Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer.

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding "molecular excited states." Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(-)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.

Biosensing Techniques↗
PubMed1999

Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit.

The waggler, a neurological mutant mouse with a disrupted putative neuronal Ca(2+) channel gamma subunit, exhibits a cerebellar granule cell-specific brain-derived neurotrophic factor deficit, severe ataxia, and impaired eyeblink conditioning. Here, we show that multiple synapses of waggler cerebellar granule cells are arrested at an immature stage during development. Synaptic transmission is reduced at parallel fiber-Purkinje cell synapses. The Golgi cell-granule cell synaptic currents show immature kinetics associated with reduced gamma-aminobutyric acid type A receptor alpha6 subunit expression in granule cells. In addition, the mossy fiber-granule cell synapses exhibit N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. Our results suggest that voltage-dependent Ca(2+) channels are involved in synapse maturation. This deficient synaptic transmission in the waggler cerebellum may account for their behavioral deficits.

Animals↗
PubMed1999

Impaired transition state complementarity in the hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases.

The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Bronsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate [Denu et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2493-2498; Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution.

Binding Sites↗
PubMed1999

Distamycin A selectively inhibits Acanthamoeba RNA synthesis and differentiation.

The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.

Acanthamoeba↗
PubMed1999

Adsorption properties and breakthrough model of 1,1-dichloro-1-fluoroethane on activated carbons.

Hydrochlorofluorocarbons (HCFCs), among the major replacements for chlorofluorocarbons (CFCs) and chlorinated solvents, are now considered to the prime contribution to the stratospheric ozone depletion. This paper reports the adsorption equilibrium of 1,1-dichloro-1-fluoroethane (HCFC-141b) vapor in air streams on the commercial activated carbons PCB and BPL, which were made from coconut shell and bituminous coal, respectively, at 283, 293, 303, and 313 K. The experimental results show that within the experimental conditions, the adsorption capacities of the two adsorbents for HCFC-141b in dry air streams can be well-fitted by the Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) adsorption equations. The physical properties of the adsorbents are consistent with the isotherm parameters obtained from the adsorption results. In addition, available experimental data of adsorption breakthrough at 283 K were used in conjunction with the Yoon and Nelson model to generate theoretical breakthrough curves based on the obtained values of parameters. In each case, calculated theoretical breakthrough curves are in agreement with corresponding experimental data.

Journal Article↗
PubMed1999

Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody.

Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT(1)( )MF-2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2-CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence-labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R-immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs.

Animals↗
PubMed1999

Apolipoprotein E promoter and alpha2-macroglobulin polymorphisms are not genetically associated with Chinese late onset Alzheimer's disease.

In this study, we investigated two newly reported polymorphisms in association with late onset Alzheimer's disease (AD) in Chinese. They were a -491 A/T polymorphism in the Apolipoprotein E (APOE) promoter region and a five base pair deletion at exon 18 of alpha2-Macroglobin (A2M). There were 196 AD and 180 normal controls (N), which were age- and sex-matched. APOE epsilon4 alleles were significantly increased in AD vs. N (chi2 = 33.3, P < 0.000001). However, neither the -491 A/T (chi2 = 1.13, P = 0.29) nor A2M (chi2 = 0.18, P = 0.67) polymorphism was associated with AD risk, suggesting that these polymorphisms do not represent risk factors for AD in the Chinese population.

Age of Onset↗
PubMed1999

The next generation of MMP inhibitors. Design and synthesis.

Since their inception during the eighties, MMP inhibitors (MMPIs) have gone through several cycles of metamorphosis. The design of early MMPIs was based on the cleavage site of peptide substrates. The second generation contained a substituted succinate scaffold (e.g., marimastat) coupled to a modified amino acid residue. The lower molecular weight analogs with multiple substitution possibilities produced a series of MMP inhibitors with varying degrees of selectivity for various MMPs. The introduction of sulfonamides in the midnineties added a new dimension to this field. The simplicity of synthesis coupled with high potency (e.g., CGS-27023A, AG-3340) produced a number of clinical candidates. This review highlights some of the key features that contributed to the discovery of this novel series of MMP inhibitors.

Caprolactam↗
PubMed1999

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

Amino Acid Sequence↗
PubMed1999

Activation of an MDM2-specific caspase by p53 in the absence of apoptosis.

Cells undergoing p53-mediated apoptosis activate caspase 3-like activities, resulting in the cleavage of the MDM2 oncoprotein and other apoptotic substrates such as poly(ADP-ribose) polymerase. To investigate the mechanism of p53-mediated apoptosis and to determine whether cleavage of MDM2 has a potential role in regulating p53, we examined caspase activation and cleavage of MDM2 in a cell line undergoing p53-mediated growth arrest and delayed apoptosis. We found that in H1299 cells expressing a temperature-sensitive human p53, a distinct caspase activity specific for the MDM2 cleavage site DVPD is induced by p53 prior to the onset of apoptosis and loss of viability. This is accompanied by the cleavage of MDM2 but not the apoptotic substrate poly(ADP-ribose) polymerase. The cleaved MDM2 loses the ability to promote p53 degradation and may potentially function in a dominant-negative fashion to stabilize p53. These results suggest that p53 activation may induce a positive feedback effect by cleavage of MDM2 through a unique caspase.

Apoptosis↗
PubMed1999

Structural basis for the binding specificity of a SSTR1-selective analog of somatostatin.

The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-[D-Trp8, IAmp9]SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-[D-Trp8, Amp9]SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2.

Animals↗
PubMed1999

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.

Amines↗
PubMed1999

Transcriptional repression of pref-1 by glucocorticoids promotes 3T3-L1 adipocyte differentiation.

Pref-1 is an epidermal growth factor-like domain-containing transmembrane protein that is cleaved to generate a soluble factor. It is abundant in 3T3-L1 preadipocytes but absent in mature adipocytes. Constitutive expression of pref-1 or the addition of its ectodomain inhibits adipogenesis. We find that the pref-1 gene is an early target of dexamethasone, a component of the dexamethasone/methylisobutylxanthine differentiation mixture used routinely for adipoconversion. The time course of the decrease in pref-1 mRNA by dexamethasone reflected the pref-1 mRNA half-life determined by actinomycin D treatment. Nuclear run-on assays showed that dexamethasone attenuates pref-1 transcription. We demonstrate a correlation between pref-1 down-regulation and adipoconversion by varying the time period and concentration of dexamethasone. Increasing the dexamethasone treatment from 2 to 4 days resulted in a time-dependent pref-1 down-regulation and increased differentiation as measured by adipocyte marker mRNAs. The dexamethasone concentration between 1 and 10 nM showed a dose-dependent decrease in pref-1 mRNA and an enhancement of adipogenesis. To test the hypothesis that dexamethasone initiation of adipoconversion may be via down-regulation of pref-1, we lowered endogenous pref-1 mRNA levels by stably transfecting 3T3-L1 preadipocytes with antisense pref-1. At 1 microM, antisense cells had enhanced adipose conversion; a similar degree of differentiation occurred with 2 nM dexamethasone, a concentration that does not support differentiation of control 3T3-L1 cells. We conclude that dexamethasone-mediated repression of pref-1 contributes to the mechanisms whereby glucocorticoids promote adipogenesis.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Lipoprotein lipase mutations and Alzheimer's disease.

Lipoprotein lipase (LPL) helps transfer lipids from lipoprotein particles to cells. In the brain, LPL is present in Alzheimer's disease (AD) amyloid plaques. LPL binds apolipoprotein E (ApoE) lipoprotein particles and low-density lipoprotein receptor-related protein (LRP), an ApoE receptor. Since polymorphisms in both ApoE and LRP influence AD risk, we sought to determine whether LPL mutations also affect AD risk. In a case-control study, the frequencies of two of the most common known LPL mutations were measured in European-Americans either clinically diagnosed or pathologically confirmed as AD or normal control (N) subjects. In clinically diagnosed subjects, the Ser447Ter mutation comprised 9.8% (62/630) of alleles in N and 3.8% (9/238) in AD, a significant difference (P = 0.0057), while the Asn291Ser mutation comprised 1.1% (5/460) of alleles in N and 5.1% (8/158) in AD, also a significant difference (P = 0.0073), though in pathologically confirmed subjects the allele frequencies for AD did not significantly differ from N for either mutation. In clinically diagnosed subjects, LPL mutations were associated with altered AD risk, suggesting a potential role for LPL in the causation of AD. Further studies in different populations should help clarify the questions raised by these results.

Aged↗
PubMed1999

Evolutionary conservation of MyoD function and differential utilization of E proteins.

The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. We have addressed this directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T12 mouse fibroblasts. We found that the ability of different homo- and heterodimers to bind DNA in vitro was an accurate measure of biological activity in vivo. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. We found that both Drosophila and chicken MyoD-related factors were able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions.

Amino Acid Sequence↗
PubMed1999

Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase.

Acetaminophen is one of the most extensively used analgesics/antipyretics worldwide, and overdose or idiopathic reaction causes major morbidity and mortality in its victims. Research into the mechanisms of toxicity and possible therapeutic intervention is therefore essential. In this study, the response of transgenic mice overexpressing human antioxidant enzymes to acute acetaminophen overdose was investigated. Animals overexpressing superoxide dismutase or plasma glutathione peroxidase demonstrated dramatic resistance to acetaminophen toxicity. Intravenous injection of glutathione peroxidase provided normal mice with nearly complete protection against a lethal dose of acetaminophen. Surprisingly, animals overexpressing intracellular glutathione peroxidase in the liver were significantly more sensitive to acetaminophen toxicity compared with nontransgenic littermates. This sensitivity appears to be due to the inability of these animals to efficiently recover glutathione depleted as a result of acetaminophen metabolism. Finally, the results suggest that glutathione peroxidase overexpression modulates the synthesis of several acetaminophen metabolites. Our results demonstrate the ability of glutathione peroxidase levels to influence the outcome of acetaminophen toxicity.

Acetaminophen↗
PubMed1999

Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage.

The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. We have found that the 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. We suggest that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. Our results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1.

Base Sequence↗