Search PubMed⌕ Search

Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 469 records · Page 26Linked to original sources
PubMed2000

[Thiol reagent thimerosal-induced Ca2+ mobilization in isolated guinea pig cochlear outer hair cells].

OBJECTIVE: To understand mechanism of cochlear outer hair cells (OHCs) intracellular Ca2+ mobilization further. METHODS: Intracellular calcium of isolated guinea pig was investigated using thimerosal, a--SH group oxidizing agent, and fura-2 fluorescence ratio imaging microscopy. RESULTS: In the presence of thimerosal, intracellular Ca2+ concentrations ([Ca2+]i) of OHCs were elevated in a dose-dependent manner. Even in Ca(2+)-free medium, Ca2+ response was still induced. The effects of thimerosal on [Ca2+]i were completely blocked and reversed by (DTT). Neither 1-100 mumol/L ryanodine nor 5-20 mmol/L caffeine altered the effects of thimerosal. Pretreatment with pertussis toxin (PTX) for 30 min did not affect the thimerosal-induced increase in [Ca2+]i The increase in [Ca2+]i when Ca2+ was added during thimerosal application in Ca(2+)-free medium was almost completely blocked by 500 mol/L LaCl3, while nifedipine did not inhibit further increase in [Ca2+]i caused by thimerosal. CONCLUSION: Oxidation of the -SH group of the OHC membrane can induce a Ca2+ release from intracellular Ca2+ stores, which are ryanodine- and caffeine-insensitive, and Ca2+ influx through non-specific Ca2+ channels, but not the nifedipine-sensitive Ca2+ Channels. The possible oxidation of--SH group gated Ca2+ channels in OHCs are worthy of further study.

Animals↗
PubMed2000

[Primary melanoma of the central nervous system].

OBJECTIVE: To investigate the status of diagnosis and treatment of primary melanoma of the central nervous system. METHODS: Six patients with primary melanoma of the central nervous system underwent surgery in our department between 1969-1999. Clinical presentation, operative findings, histological diagnosis, and prognosis were reviewed. RESULTS: No typical symptom occurred in these patients. The lesions can mimic meningiomas and acoustic neuromas in location and radiological appearance. Total removal of tumors was only achieved in one case. Histological examination showed malignant lesions in 3 cases and benign lesions in others. Five cases were followed up and only two were alive. CONCLUSIONS: Primary melanomas are extremely rare tumors of the central nervous system. Surgical removal of the lesion may result in prolonged remission or cure.

Adolescent↗
PubMed2000

[Immobilization and properties of angiotensin converting enzyme].

OBJECTIVE: To study the immobilization of angiotensin converting enzyme (ACE) for purifying ACE inhibitor from a native peptide mixture. METHODS: The experiment was carried out under the low water activity condition, using tosylate chloride activating side-chain hydroxyl group of Sepharose CL-4B agarose to form a high active group which could react with the free amino-group of ACE to link the enzyme with agarose. RESULTS: Immobilized ACE not only had a wider pH range, but also had a 0.6 unit right-move optimum pH than soluble ACE. After treated in pH9.0 and pH 6.5 conditions for 24 h respectively, 82% and 68% enzyme activities of immobilized ACE was maintained, and soluble ACE remained 64% and 39%. Immobilized and soluble ACE both appeared maximum enzyme activity at about 50 degrees C, the soluble ACE would lose almost all its activity when temperature kept rising. When kept at 40 degrees C and 50 degrees C for 2 h, the activity of immobilized ACE remained 82% and 34% respectively, while the soluble ACE remained 52% and completely inactivated. After two kinds of enzymes were stored at 20 degrees C for one month, immobilized ACE remained 61% activity, as compared with the 20% activity residual of soluble ACE. CONCLUSION: The immobilized ACE had a better stability than soluble ACE in conditioned pH and temperature.

Enzyme Stability↗
PubMed2000

[Assay and L1 gene sequence analysis of human papillomavirus type 6 and 11 in condylomata acuminata].

OBJECTIVE: To determinate the prevalence of HPV type 6, 11 in condylomata acuminata, and analyze the sequence of L1 gene and its deduced L1 protein. METHODS: PCR was used to amplify DNA sequences between E6 and E7 of HPV type 6 and 11. The PCR products were identified by the fragment length cleaved with restriction enzyme RsaI. L1 genes were amplified with high fidelity Taq, and cloned DNA sequences were detected by the dideoxynucleotide method. RESULTS: In 34 examples of condylomata acuminata, HPV11 was found in 25 examples (73.5%), HPV6 in 15(44.1%), including HPV6 + HPV11 in 6(17.6%). In comparison with the prototypes there were 7-8 point mutations in HPV11 L1 gene and 3 in HPV6L1 gene. L1 encoded protein changes showed up only in 1-3 amino acids in the former cases. CONCLUSION: Most of patients tested with condylomata acuminata are mainly infected by HPV11, the rest of them by HPV6 or by co-infection of two mixex types. Sequence analysis of L1 genes indicated that mutations were found in both HPV6 and 11, whereas deduced amino acid mutations in L1 protein were limited in HPV11 only.

Adult↗
PubMed2000

[Determination of trace amounts of nitrite and reaction mechanism by triple-wavelength spectrophotometry].

A new method was proposed for the determination of nitrite ion by triple-wavelength spectrophotometry. This limits ranges of determination are 0-0.560 mg.L-1 nitrite ion at 599.1 nm, 420.6 nm and 283.4 nm. It has been applied to analysis trace amounts of nitrite ion of standard sample and environmental water with satisfactory result, the relative error was less 1.7% and recovery of the real samples was between 96.5 and 100.0%. This method is based on the diazotization-coupling react of thionine with nitrite ion in acidic medium, the mole ratio of thionine:nitrite is 2:1 by the continuous variation and mole ratio method.

Nitrites↗
PubMed1999

The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity.

The chaperonin GroEL is a double toriodal assembly that with its cochaperonin GroES facilitates protein folding with an ATP-dependent mechanism. Nonnative conformations of diverse protein substrates bind to the apical domains surrounding the opening of the double toroid's central cavity. Using phage display, we have selected peptides with high affinity for the isolated apical domain. We have determined the crystal structures of the complexes formed by the most strongly bound peptide with the isolated apical domain, and with GroEL. The peptide interacts with the groove between paired alpha helices in a manner similar to that of the GroES mobile loop. Our structural analysis, combined with other results, suggests that various modes of molecular plasticity are responsible for tight promiscuous binding of nonnative substrates and their release into the shielded cis assembly.

Chaperonin 10↗
PubMed1999

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels.

The G protein-coupled inward rectifier K(+) channel (GIRK) is activated by direct interaction with the heterotrimeric GTP-binding protein betagamma subunits (Gbetagamma). However, the precise role of Gbeta and Ggamma in GIRK activation remains to be elucidated. Using transient expression of GIRK1, GIRK2, Gbeta1, and Ggamma2 in human embryonic kidney 293 cells, we show that C-terminal mutants of Gbeta1, which do not bind to Ggamma2, are still able to associate with GIRK, but these mutants are unable to induce activation of GIRK channels. In contrast, other C-terminal mutants of Gbeta1 that bind to Ggamma2, are capable of activating the GIRK channel. These results suggest that Ggamma plays a more important role than that of an anchoring device for the Gbetagamma-induced GIRK activation.

Cell Line↗
PubMed1999

Phosphorylation uncouples the gastrin-releasing peptide receptor from G(q).

Previous work on the desensitization of G protein-coupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G(q)-coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of approximately 1 mol PO(4)/mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V(max)) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the alpha(q) or betagamma subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.

Animals↗
PubMed1999

The mass spectral fragmentation of 1a,3-disubstituted 4-benzoyl-1a,2,3,4- tetrahydrooxazirino

The mass spectrometric behaviour of four 1a,3-disubstituted 4-benzoyl-1a,2,3,4-tetrahydrooxazirino[2,3-a][1,5]benzodiazepines has been studied with the aid of mass-analyzed ion kinetic energy spectrometry and accurate mass measurements under electron impact ionization. All compounds show a tendency to eliminate a neutral oxygen atom, or oxygen atom plus benzoyl or aryl radicals, or oxygen atom plus CO molecule to yield 2,3-dihydro-1H-1,5-benzodiazepine derivative ions, or 1-benzoyl-2,3-dihydro-1H-1,5-benzodiazepine ions, which could further lose benzoyl to give 2,3-dihydro-1H-1,5-benzodiazepine ions. All compounds also show a tendency to eliminate a benzoyl radical to produce 1a,2,3,4-tetrahydrooxazirino[2,3-a][1,5]benzodiazepine ions. Both oxazirino[2,3-a][1,5]benzodiazepine ions and 2,3-dihydro-1H-1,5-benzodiazepine ions can undergo diazepine ring contraction rearrangement to yield benzimidazole ions by loss of propene or styrenes and other small fragments. The oxazirino[2,3-a][1,5]benzodiazepine ions [M(+)-PhCO] also undergo rearrangement reactions to form benzoxazole ions and benzimidazole ions. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed1999

Chemical syntheses and biological studies on dimeric chimeras of oxytocin and the V(2)-antagonist, d(CH(2))(5)[D-Ile(2), Ile(4)]arginine vasopressin.

Parallel and antiparallel heterodimers have been synthesized that combine into a single molecule the neurohypophyseal hormone oxytocin and the potent vasopressin V(2)-antagonist d(CH(2))(5)[D-Ile(2), Ile(4)]arginine vasopressin. Solid-phase synthesis with N(alpha)-9-fluorenylmethyloxycarbonyl (Fmoc) chemistry, featuring appropriate combinations of orthogonal protecting groups for the thiols [S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm); S-acetamidomethyl (Acm); S-triphenylmethyl (Trt)], was used to assemble the required linear nonapeptide amide monomer intermediates, which were then brought together in defined ways by solution reactions to provide the two heterodimers. The first disulfide bridge was formed by a directed approach involving attack by the free thiol of the 1-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid (Pmp) residue of one monomer onto the Snm group of a cysteine residue on the other monomer; the inverse directed strategy failed due to steric hindrance. The second disulfide bridge was formed by iodine co-oxidation of Cys(Acm) residues on adjacent chains. Biological studies revealed that both the parallel and antiparallel chimeras lack pressor activity, have low uterotonic activity, and have diuretic activities comparable to that of the monomeric V(2)-antagonist. Sodium excretion depends on experimental conditions. Thus, with a 4% water load, both chimeras display effects similar to that of an equimolar mixture of oxytocin and V(2)-antagonist, i.e., lower sodium excretion than that resulting from administration of oxytocin alone but higher than that when V(2)-antagonist was administered alone. However, when no water load was used, the parallel chimera proved to be more effective in promoting sodium excretion than either oxytocin alone or an equimolar mixture of oxytocin and V(2)-antagonist.

Amino Acid Sequence↗
PubMed1999

The complete DNA sequence of myxoma virus.

Myxomatosis in European rabbits is a severely debilitating disease characterized by profound systemic cellular immunosuppression and a high rate of mortality. The causative agent, myxoma virus, is a member of the poxvirus family and prototype of the Leporipoxvirus genus. As a major step toward defining the genetic strategies by which the virus circumvents host antiviral responses, the genomic DNA sequence of myxoma virus, strain Lausanne, was determined. A total of 171 open reading frames were assigned to cover the 161.8-kb genome, including two copies each of the 12 genes that map within the 11.5-kb terminal inverted repeats. Database searches revealed a central core of approximately 120 kb that encodes more than 100 genes that exhibit close relationships to the conserved genes of members of other poxvirus genera. Open reading frames with predicted signal sequences, localization motifs, or homology to known proteins with immunomodulatory or host-range functions were examined more extensively for predicted features such as hydrophobic regions, nucleic acid binding domains, ankyrin repeats, serpin signatures, lectin domains. and structural cysteine spacings. As a result, several novel, potentially immunomodulatory proteins have been identified, including a family with multiple ankyrin-repeat domains, an OX-2 like member of the neural cell adhesion molecule family, a third myxoma serpin, a putative chemokine receptor fragment, two natural killer receptor-like species, and a variety of species with domains closely related to diverse host immune regulatory proteins. Coupled with the genomic sequencing of the related leporipoxvirus Shope fibroma virus, this work affirms the existence of a conserved complement of poxvirus-specific core genes and expands the growing repertoire of virus genes that confer the unique capacity of each poxvirus family member to counter the immune responses of the infected host.

Amino Acid Sequence↗
PubMed1999

Characterization of TNFRSF19, a novel member of the tumor necrosis factor receptor superfamily.

By searching the expressed sequence tag database, a novel murine tumor necrosis factor receptor designated TNFRSF19 was identified. TNFRSF19 cDNA encodes a putative membrane protein of 348 amino acids with one incomplete and two complete cysteine-rich motifs within its extracellular region and a large cytoplasmic domain. TNFRSF19 mRNA can be detected in most murine tissues examined, particularly in brain, reproductive organs, and late developmental stages of murine embryo, but not in tissues of the immune system. The cell surface expression of the ligand of TNFRSF19 is highly restricted. Of 22 human and murine cell lines examined by FACS analysis, only Raji (B cell lymphoma cell line), GM847 (fibroblast cell line), 293 (embryonic kidney cell line), and K562 (chronic myeloid leukemia) were positive. TNFRSF19 did not bind newly cloned TNF ligands, including TWEAK (HGMW-approved symbol TNFSF12), VEGI/TL1 (HGMW-approved symbol TNFSF15), TL6/endokine (HGMW-approved symbol TNFSF18), APRIL (HGMW-approved symbol TNFSF13), OPGL (HGMW-approved symbol TNFSF11), LIGHT (HGMW-approved symbol TNFSF14), or BAFF/THANK (HGMW-approved symbol TNFSF13B) by enzyme-linked immunosorbent assay and FACS analyses. Overexpression of TNFRSF19 transduced neither apoptotic signaling nor signals leading to NF-kappaB induction. Taken together with the data that the TNFRSF19 extracellular domain-immunoglobulin fusion protein did not affect the allogeneic mixed lymphocyte reaction, our data indicate that TNFRSF19 is not involved in the modulation of immune responses.

Amino Acid Sequence↗
PubMed1999

Bastadin 10 stabilizes the open conformation of the ryanodine-sensitive Ca(2+) channel in an FKBP12-dependent manner.

The marine sponge Ianthella basta synthesizes at least 25 tetrameric bromotyrosine structures that possess a stringent structural requirement for modifying the gating behavior of ryanodine-sensitive Ca(2+) channels (ryanodine receptors) (RyR)). Bastadin 5 (B5) was shown to stabilize open and closed channel states with little influence on the sensitivity of the channel to activation by Ca(2+) (Mack, M. M., Molinski, T. F., Buck, E. D., and Pessah, I. N. (1994) J. Biol. Chem. 269, 23236-23249). In the present paper, we utilize single channel analysis and measurements of Ca(2+) flux across the sarcoplasmic reticulum to identify bastadin 10 (B10) as the structural congener responsible for dramatically stabilizing the open conformation of the RyR channel, possibly by reducing the free energy associated with closed to open channel transitions (DeltaG*c --> o). The stability of the channel open state induced by B10 sensitized the channel to activation by Ca(2+) to such an extent that it essentially obviated regulation by physiological concentrations of Ca(2+) and relieved inhibition by physiological Mg(2+). These actions of B10 were produced only on the cytoplasmic face of the channel, were selectively eliminated by pretreatment of channels with FK506 or rapamycin, and were reconstituted by human recombinant FKBP12. The actions of B10 were found to be reversible. A structure-activity model is proposed by which substitutions on the Eastern and Western hemispheres of the bastarane macrocycle may confer specificity toward the RyR1-FKBP12 complex to stabilize either the closed or open channel conformation. These results indicate that RyR1-FKBP12 complexes possesses a novel binding domain for phenoxycatechols and raise the possibility of molecular recognition of an endogenous ligand.

Adenosine Triphosphate↗
PubMed1999

Dopamine transporter mRNA in autopsy studies of chronic cocaine users.

The effects of chronic cocaine abuse on transcriptional regulation of human dopamine transporter (DAT) mRNA in midbrain dopaminergic neurons was assessed by reverse transcription-polymerase chain reaction (RT-PCR). DAT/cyclophilin mRNA ratios in the substantia nigra (SN) were unchanged in cocaine overdose (CO) victims as compared to age-matched and drug-free control (CTRL) subjects. In contrast, DAT mRNA levels were decreased significantly in agitated cocaine delirium victims (67%, p<0.05). These findings demonstrate the lack of a regulatory influence by cocaine on the steady state content of DAT mRNA in cocaine abusers. In contrast, DAT gene expression was altered significantly in cocaine abusers at risk for agitated delirium.

Adult↗
PubMed1999

Myoglobin-containing carbon-paste enzyme microelectrodes for the biosensing of glucose under oxygen-deficit conditions.

The response of first-generation glucose oxidase (GOx) amperometric glucose biosensors is strongly dependent on the concentration of the oxygen cosubstrate. The incorporation of the natural oxygen binder myoglobin into a GOx-containing carbon-paste matrix is shown to satisfy the oxygen demand of the enzymatic reaction and to provide convenient biosensing of glucose in oxygen-free solutions. Such use of myoglobin-containing mineral oil thus offers an attractive alternative to the use of oxygen-rich fluorocarbon pasting liquids. Further improvements are observed upon doping the fluorocarbon oil with myoglobin. Factors affecting the oxygen independence of the new enzyme microelectrodes, including the myoglobin loading or length of the oxygen reservoir, have been optimized. The myoglobin-doped mineral oil or Kel-F-based carbon-paste enzyme microelectrodes display a highly stable glucose response over prolonged (6-7 h) operations in oxygen-free solutions, indicating no depletion of the internal oxygen supply.

Biosensing Techniques↗
PubMed1999

Oligodeoxynucleotides containing CpG motifs can induce rejection of a neuroblastoma in mice.

Phosphorothioate oligodeoxynucleotides with CpG motifs (CpG-ODNs) activate various immune cell subsets and induce production of numerous cytokines. To evaluate whether CpG-ODNs can induce rejection of established malignant tumor, A/J mice were challenged by the s.c. implantation of a syngenic neuroblastoma cell line (neuro2a) and subsequently injected with CpG-ODNs in the vicinity of the tumor. Daily injections of 10 microg CpG-ODNs for 15 days seemed to be the most potent regimen, leading to the eradication of 5-mm-diameter tumors in one-half of the animals and a significant tumor growth inhibition when compared with controls (88% reduction volume; P<0.001). CpG-ODN-cured animals were further protected against a new tumor challenge. The antitumoral effect of CpG-ODNs was dependent on CpG motifs, and natural killer cells seemed to play a critical role in tumor rejection. We conclude that immunostimulatory CpG-ODNs may induce the rejection of established tumors and warrant further evaluation as a potential immunotherapeutic agent.

Adjuvants, Immunologic↗
PubMed1999

Crystal structure of the stromelysin catalytic domain at 2.0 A resolution: inhibitor-induced conformational changes.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.

Allosteric Regulation↗