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Biomedical subjects

G Chen

Publications and source records attributed to G Chen.

At least 235 records · Page 13Linked to original sources
PubMed2000

ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999.

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Galpha(i) have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Galpha(i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca(2+) mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Galpha subunits (Galpha(q/i)). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC(50) of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, Galpha(i)-linked receptor for ADP.

3T3 Cells↗
PubMed2000

The C. elegans orthologue ceBNIP3 interacts with CED-9 and CED-3 but kills through a BH3- and caspase-independent mechanism.

We have studied ceBNIP3, the orthologue of BNIP3 in C. elegans. Sequence analysis reveals that the different domains of BNIP3 have been conserved throughout evolution. ceBNIP3 contains a C-terminal transmembrane (TM) domain, a conserved domain (CD) of 19 amino acids, a BCL-2 homology-3 (BH3)-like domain and a PEST sequence. ceBNIP3 is expressed primarily as a 25 kDa monomer and a 50 kDa homodimer. After transfection, ceBNIP3 protein is rapidly degraded through a ubiquitin-dependent pathway by the proteasome. Like BNIP3, the TM domain of ceBNIP3 mediates the localization of the protein to mitochondria and is also necessary for homodimerization and cell death in mammalian cells. Neither the putative BH3 domain nor conserved domain is necessary for killing. ceBNIP3 protein interacts with CED-9 and BCL-XL, but unlike other pro-apoptotic BCL-2 family members, the BH3-like domain does not participate in dimerization. The ceBNIP3 TM domain mediates interaction with both CED-9 and BCL-XL. ceBNIP3 interacts with CED-3 but co-expression of CED-3 and ceBNIP3 does not significantly enhance induction of cell death in the presence or absence of CED-4. ceBNIP3 kills mammalian cells by a caspase-independent mechanism. In conclusion, we find that although ceBNIP3 interacts with CED-9 and CED-3 it kills by a BH3- and caspase-independent mechanism.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.

Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution.

Amino Acid Sequence↗
PubMed2000

Clinical and preclinical evidence for the neurotrophic effects of mood stabilizers: implications for the pathophysiology and treatment of manic-depressive illness.

Recent neuroimaging studies have demonstrated regional central nervous system volume reductions in mood disorders, findings that are complemented by postmortem observations of cell atrophy and loss. It is thus noteworthy that lithium and valproate have recently been demonstrated to robustly increase the expression of the cytoprotective protein bcl-2 in the central nervous system. Chronic lithium not only exerts neuroprotective effects in several preclinical paradigms but also enhances hippocampal neurogenesis. Valproate robustly promotes neurite outgrowth and activates the ERK mitogen-activated protein kinase pathway, a signaling pathway utilized by many endogenous neurotrophic factors. Consistent with its preclinical neurotrophic/neuroprotective effects, chronic lithium treatment of patients with manic-depressive illness increases brain N-acetylaspartate (a putative marker of neuronal viability and function) levels, an effect that is localized almost exclusively to gray matter. To determine if lithium was producing neuropil increases, quantitative three-dimensional magnetic resonance imaging studies were undertaken, which revealed that chronic lithium significantly increases total gray matter volume in the human brain of patients with manic-depressive illness. Together, these results suggest that a reconceptualization about the optimal long-term treatment of recurrent mood disorders is warranted. Optimal long-term treatment for these severe illnesses may only be achieved by the early use of agents with neurotrophic/neuroprotective effects, irrespective of the primary, symptomatic treatment.

Affect↗
PubMed2000

Lithium-induced increase in human brain grey matter.

Rodent studies have shown that lithium exerts neurotrophic or neuroprotective effects. We used three-dimensional magnetic resonance imaging and brain segmentation to study pharmacologically-induced increases in grey matter volume with chronic lithium use in patients with bipolar mood disorder. Grey-matter volume increased after 4 weeks of treatment. The increases in grey matter probably occurred because of neurotrophic effects.

Adolescent↗
PubMed2000

Optically induced entanglement of excitons in a single quantum Dot

Optically induced entanglement is identified by the spectrum of the phase-sensitive homodyne-detected coherent nonlinear optical response in a single gallium arsenide quantum dot. The electron-hole entanglement involves two magneto-excitonic states differing in transition energy and polarization. The strong coupling needed for entanglement is provided through the Coulomb interaction involving the electrons and holes. The result presents a first step toward the optical realization of quantum logic operations using two or more quantum dots.

Journal Article↗
PubMed2000

Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells.

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2alphabeta, hence is designated as MIP-2gamma. Mouse MIP-2gamma was identified by electrocloning and is highly homologous to human MIP-2gamma. Northern blotting revealed that MIP-2gamma was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2gamma was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2gamma expression by monocyte-derived DC. Recombinant MIP-2gamma from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2gamma is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2gamma does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

Adult↗
PubMed2000

Concurrent measures of protein kinase C and phosphoinositides in lithium-treated bipolar patients and healthy individuals: a preliminary study.

This study examined the hypothesis that lithium inhibits the PI signaling pathway in humans during in vivo administration by concurrently measuring PKC isozymes and platelet membrane phosphoinositides in lithium-treated patients and healthy individuals. The platelet membrane and cytosolic levels of PKC alpha, beta I, beta II, delta, and epsilon were measured using Western blotting. The relative platelet membrane contents of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP(2)) were measured with two-dimensional thin-layer chromatography. Nine euthymic lithium-treated bipolar subjects and 11 healthy control subjects were studied. Compared to control subjects, lithium-treated bipolar patients had significantly lower levels of cytosolic PKC alpha isozyme (t-test=-3.24, d.f.=17, P=0.01) and PIP(2) platelet membrane levels (t-test=-2.51, d.f.=18, P=0.02), and a trend toward reduced levels of cytosolic PKC beta II isozyme (t=-2.17, d.f.=17, P=0.05). There was no significant correlation between PIP(2) and any of the PKC isozymes. These preliminary findings suggest that chronic lithium treatment may decrease the levels of both cytosolic PKC alpha isozyme and membrane PIP(2) in platelets of bipolar disorder patients.

Adult↗
PubMed2000

Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.

TR3, an immediate-early response gene and an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily of transcription factors, regulates apoptosis through an unknown mechanism. In response to apoptotic stimuli, TR3 translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis. Mitochondrial targeting of TR3, but not its DNA binding and transactivation, is essential for its proapoptotic effect. Our results reveal a mechanism by which a nuclear transcription factor translocates to mitochondria to initiate apoptosis.

Apoptosis↗
PubMed2000

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy.

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Amino Acid Sequence↗
PubMed2000

Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells.

Chinese hamster ovary cell production of recombinant tissue-type plasminogen activator (t-PA) was increased by amplification of cotransfected dihydrofolate reductase cDNA using stepwise adaptation to increasing methotrexate (MTX) concentrations. The highest producing clones were isolated at 5 microM MTX and yielded 26,000 U/10(6) cells/day t-PA (43 microgram/10(6) cells/day). Above 25 microM MTX, cell specific t-PA production rates became increasingly variable and the cDNA copynumbers decreased. No apparent correlation between the cell specific t-PA production rate and the growth rate was observed upon subcloning of the amplified cells. When MTX selection was removed, the t-PA production rate decreased up to tenfold within 40 days; this was accompanied by an up to 60% drop in cDNA copynumber. Subclones isolated after 108 days of culture in the absence of MTX were, on average, sixfold more stable than their parental cells. In culture without MTX, the maximum stable t-PA production rate obtained (over 250 days) was 7000 +/- 750 U/10(6) cells/day (approximately 12 microgram/10(6) cells/day), approximately threefold lower than the maximum unstable levels of production reached under selective pressure. Taken together, these results define a wide range of the highest t-PA expression rates obtained under MTX selection, for which stable expression without selection has not been reported.

Animals↗
PubMed2000

Lithium increases N-acetyl-aspartate in the human brain: in vivo evidence in support of bcl-2's neurotrophic effects?

BACKGROUND: Recent preclinical studies have shown that lithium (Li) robustly increases the levels of the major neuroprotective protein, bcl-2, in rat brain and in cells of human neuronal origin. These effects are accompanied by striking neuroprotective effects in vitro and in the rodent central nervous system in vivo. We have undertaken the present study to determine if lithium exerts neurotrophic/ neuroprotective effects in the human brain in vivo. METHODS: Using quantitative proton magnetic resonance spectroscopy, N-acetyl-aspartate (NAA) levels (a putative marker of neuronal viability and function) were investigated longitudinally in 21 adult subjects (12 medication-free bipolar affective disorder patients and 9 healthy volunteers). Regional brain NAA levels were measured at baseline and following 4 weeks of lithium (administered in a blinded manner). RESULTS: A significant increase in total brain NAA concentration was documented (p < .0217). NAA concentration increased in all brain regions investigated, including the frontal, temporal, parietal, and occipital lobes. CONCLUSIONS: This study demonstrates for the first time that Li administration at therapeutic doses increases brain NAA concentration. These findings provide intriguing indirect support for the contention that chronic lithium increases neuronal viability/function in the human brain, and suggests that some of Li's long-term beneficial effects may be mediated by neurotrophic/neuroprotective events.

Adult↗
PubMed2000

Separate domains in E1 and E2 proteins serve architectural and productive roles for cooperative DNA binding.

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to binding sites in the viral origin of DNA replication. The DNA-binding domains (DBDs) of the two proteins interact with each other, and the E2 transactivation domain interacts with the helicase domain of E1. Mutations that disrupt the interaction between the two DBDs also disrupt the interaction between the E2 activation domain and the E1 helicase domain, demonstrating interdependence of the two interactions. Cooperative binding of the two DBDs generates a sharp bend in the DNA that is required for interaction between the E2 activation domain and E1. This indicates that interaction between the two DBDs plays an architectural role, 'triggering' a productive interaction between the E2 transactivation domain and E1 through introduction of a sharp bend in the DNA. This two-step mechanism may be a required feature for cooperative DNA binding to proximal binding sites.

Binding Sites↗
PubMed2000

Purification of human prothrombin from Nitschmann fraction III using DEAE membrane radial flow chromatography.

A DEAE membrane radial column (4x5 cm) was used to purify human prothrombin from Nitschmann fraction III and performances of the column were investigated. Sample flow-rate of 20-30 ml/min (i.e., 24-36 bed volumes/h) and elution velocity up to 40 ml/min (i.e., 48 bed volumes/h) were obtained without compromising the separation efficiency. Its breakthrough capacity was one-third to half that of the commercial DEAE Sepharose Fast Flow media. It was concluded that the novel column is an attractive alternate to traditional axial column.

Chromatography, Liquid↗
PubMed2000

Groucho/TLE family proteins and transcriptional repression.

The Drosophila Groucho (Gro) protein is the prototype for a large family of corepressors, examples of which are found in most metazoans. This family includes the human transducin-like Enhancer of split (TLE) proteins. As corepressors, Gro/TLE family proteins do not bind to DNA directly, but rather are recruited to the template by DNA-bound repressor proteins. Gro/TLE family proteins are required for many developmental processes, including lateral inhibition, segmentation, sex determination, dorsal/ventral pattern formation, terminal pattern formation, and eye development. These proteins are characterized by a conserved N-terminal glutamine-rich domain and a conserved C-terminal WD-repeat domain. The primary role of the glutamine-rich domain is apparently to mediate tetramerization, while the WD-repeat domain may mediate interactions with DNA-bound repressors. The glutamine rich and WD-repeat domains are separated by a less conserved region containing domains that have been implicated in transcriptional repression and nuclear localization. In addition to encoding full-length Gro/TLE family proteins, most metazoan genomes encode truncated family members that contain the N-terminal oligomerization domain, but lack the C-terminal WD-repeat domain. These truncated proteins may negatively regulate full-length Gro/TLE proteins, perhaps by sequestering them in non-productive complexes. Gro/TLE family proteins probably repress transcription by multiple mechanisms. For example, a glycine/proline-rich domain in the central variable region functions to recruit the histone deacetylase Rpd3 to the template. This histone deacetylase then presumably silences transcription by altering local chromatin structure. Other repression domains in Gro may function in a histone deacetylase-independent manner. Many aspects of Gro/TLE protein function remain to be explored, including the possible post-translational regulation of Gro/TLE activity as well as the mechanisms by which Gro/TLE proteins direct repression at a distance.

Animals↗
PubMed2000

4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7.

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.

Glucuronides↗