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L Chen

Publications and source records attributed to L Chen.

At least 793 records · Page 44Linked to original sources
PubMed1997

Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc.

Transcription factors of the NFAT family regulate the production of effector proteins that coordinate the immune response. The immunosuppressive drugs FK506 and cyclosporin A (CsA) act by blocking a Ca2+-mediated signalling pathway leading to NFAT. Although FK506 and CsA have enabled human organs to be transplanted routinely, the toxic side-effects of these drugs limit their usage. This toxicity might be absent in antagonists that target NFAT directly. As a first step in the structure-based search for NFAT antagonists, we now report the identification and solution structure of a 20K domain of NFATc (NFATc-DBD) that is both necessary and sufficient to bind DNA and activate transcription cooperatively. Although the overall fold of the NFATc DNA-binding domain is related to that of NF-kappaB p50 (refs 2, 3), the two proteins use significantly different strategies for DNA recognition. On the basis of these results, we present a model for the cooperative complex formed between NFAT and the mitogenic transcription factor AP-1 on the interleukin-2 enhancer.

Amino Acid Sequence↗
PubMed1997

TGF-alpha, EGF, and their cognate EGF receptor are co-expressed with desmin during embryonic, fetal, and neonatal myogenesis in mouse tongue development.

The developing mouse tongue provides a model for discrete patterns of morphogenesis during short periods of embryonic development. Occipital somite-derived myogenic cells interact with cranial neural crest-derived ecto-mesenchymal cells to form the musculature of the tongue. The biochemical signals that control close range autocrine and/or paracrine signaling processes required to establish the fast-twitch complex tongue musculature are not known. The present study was designed to test the hypothesis that desmin, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF alpha) and their cognate receptor, epidermal growth factor receptor (EGFr), are co-expressed during tongue myogenesis and define specific developmental stages of tongue muscle cell differentiation. To test this hypothesis, we performed studies to analyze the timing, position, and concentration of desmin, TGF alpha, EGF, and EGFr from embryonic day 9 (E9) through birth in Swiss Webster mouse tongue development. Desmin, TGF alpha, EGF, and EGFr co-localized to cells of myogenic lineage in the four occipital somites and subsequently in myoblasts and myotubes from E9 through E17. By newborn stage, desmin is localized to discrete regions in myofibers corresponding to Z-line delimiting sarcomeres, and A-band within sarcomeres; immunostaining for desmin, TGF alpha, and EGF persisted in differentiated myotubes and striated skeletal muscle. Desmin increased from 0.01% at E11 to 0.51% of the total protein by E17 and at birth. Concomitantly, the patterns and increases in TGF alpha, EGF, and EGFr showed significant increases during the same developmental period. The temporal and positional co-localization of TGF alpha, EGF, and EGFr support the hypothesis that autocrine and paracrine regulation of desmin by actions of growth factor ligand and receptor defines critical stages of tongue myogenesis.

Animals↗
PubMed1997

Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules.

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.

Base Sequence↗
PubMed1997

Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions.

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure.

Amino Acid Sequence↗
PubMed1997

Effects of phenethyl isothiocyanate on acetaminophen metabolism and hepatotoxicity in mice.

Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous and other vegetables, is a potent inhibitor of cytochrome P450 2E1. This enzyme catalyzes the bioactivation of acetaminophen (APAP) and many other xenobiotics. The present study investigated the effects of PEITC on APAP metabolism and associated hepatotoxicity in Swiss-Webster mice. When PEITC (19-150 micromol/kg) was given to mice intragastrically 1 hr before or immediately prior to a toxic dose of APAP, the APAP-induced hepatotoxicity was significantly decreased or was completely prevented. The extent of toxicity was evaluated by mortality, serum levels of glutamic-pyruvic transaminase, lactate dehydrogenase, and liver histopathology. Pretreatment of mice with ethanol enhanced APAP hepatotoxicity; this enhanced toxicity could also be prevented by the administration of PEITC. PEITC treatment prevented the depletion of hepatic glutathione levels caused by oxidized APAP metabolites. PEITC treatment also significantly decreased the plasma levels of oxidized APAP metabolites (analyzed as APAP-glutathione, APAP-cysteine, and APAP-N-acetylcysteine) and reduced the urinary excretion of APAP-cysteine. In microsomal incubations, PEITC effectively inhibited the rate of APAP-glutathione formation from APAP as well as the P450 2E1-dependent N-nitrosodimethylamine demethylase and the P450 1A2-dependent ethoxyresorufin O-deethylase activities. The protective action of PEITC against APAP toxicity is attributed to the blocking of APAP activation through inhibition of P450 enzymes.

Acetaminophen↗
PubMed1997

Transduction of murine and human tumors using recombinant adenovirus vectors.

BACKGROUND: Most cytokine-based cancer gene therapy clinical trials have used labor-intensive, retrovirus-mediated strategies resulting in unpredictable gene expression. Recombinant AdV vectors were evaluated for easier, more reproducible gene transfer into 12 human melanoma, 2 murine fibrosarcomas, and 8 other tumor cell lines. METHODS: AdV vectors contained a reporter (Escherichia coli beta-galactosidase or firefly luciferase) or cytokine gene (human interleukin-2 [IL-2] or IL-7). Transduction efficiencies and expression levels were assessed by histochemical staining, flow cytometry, polymerase chain reaction, fluorometry, and enzyme-linked immunosorbent assay. Tumorigenicity was determined by subcutaneous injection of cells into syngeneic mice. RESULTS: All cell lines studied were transduced with AdV. Most cell lines exhibited 100% transduction efficiencies (by flow cytometry) at multiplicities of infection (MOI) epsilon 10. Gene expression correlated linearly with MOI, but a cytopathic effect was observed at MOI > 100 with all vectors. Nanogram gene expression levels were routinely achieved. Irradiation (30 Gy) minimally affected expression levels. Tumorigenicity of AdV-IL-2-transduced fibrosarcoma cells in mice was inversely related to IL-2 production. A majority of mice that rejected their tumor challenge were immune to tumor rechallenge. CONCLUSIONS: E1-deleted AdV vectors may prove useful in generating tumor vaccines ex vivo with high, transient cytokine expression levels.

Adenoviridae↗
PubMed1997

Upstream sequencing and functional characterization of the human cholinergic gene locus.

The 5' flanking region of the human VAChT gene was sequenced to approx 5350 bases upstream of the initiating methionine codon of the VAChT open reading frame (orf). The 5' flanks of the human and rat cholinergic gene loci were compared to identify regions of local sequence conservation, and therefore of potential regulatory importance. Several discrete domains of high homology, including a cluster of far-upstream cis-active consensus motifs, a neuronally restrictive silencer element consensus sequence, and additional conserved sequences within the putative nerve growth factor response domain of the locus, were identified. The probable start of transcription of the VAChT gene was deduced from mapping of sequences of rat and human VAChT cDNAs onto the 5' flanking regions of the human and rat cholinergic gene loci. The actual utilization of a putative 5' VAChT exon in rat central nervous system (CNS) tissue was assessed by in situ hybridization histochemistry. RNA transcripts containing both VAChT and ChAT protein-coding sequences were abundant in spinal cord motoneurons, sympathetic preganglionic cells, basal forebrain, striatum, and cranial motor nuclei. R-exon-containing transcripts could be detected only at low levels in these cell groups, implying that most transcription of VAChT proceeds from a promoter downstream of the R-exon. To assess the structural requirements for expression of the VAChT gene without bias regarding the actual start of transcription, a 5' fragment of the human gene corresponding to approximately 3 kb of sequence extending upstream from within the presumed 5' untranslated region of VAChT itself was fused to a luciferase-encoding reporter and transfected into VAChT-expressing and nonexpressing human and rat cell lines. This portion of the VAChT gene provided strong promoter expression in both cholinergic and noncholinergic cell lines. Deletion of the putative neuronally restrictive silencer element (NRSE) resulted in enhanced transcription in all cell lines. Lack of differential expression of VAChT transcription in VAChT-expressing vs non-VAChT-expressing cell lines suggested that additional enhancer elements controlling cell-specific expression of the VAChT gene exist further upstream in the cholinergic locus 5' flank. Conservation of potential cis-active elements within a 1.4 kb sequence immediately upstream of the NRSE in both rat and human cholinergic gene loci suggests that this domain is required for cholinergic-specific regulation of VAChT and ChAT gene transcription.

Acetylcholine↗
PubMed1997

Induction of osteocalcin gene expression in vitro by progesterone.

The effect of progesterone on osteocalcin gene expression was investigated in order to understand how progesterone acts on osteoblast in vitro. For this purpose, fetal rat calvarial osteoblasts were cultured in the medium containing 10(-9) M-10(-6) M progesterone respectively for two weeks while the cells progressing differentiation. Northern blot analysis showed that progesterone increased the level of osteocalcin mRNA in a dose-dependent manner. Among those doses of progesterone employed in this study, only 10(-6) M hormone caused a significant induction of osteocalcin gene expression (2.3 fold, P < 0.01). Our results demonstrated for the first time that the osteocalcin gene expression is regulated by progesterone. In addition, the results obtained from our study provides a good evidence for the direct effect of progesterone on osteoblast in vitro.

Animals↗
PubMed1997

The effect of levobunolol hydrochloride on the calcium and potassium channels in isolated ventricular myocytes of guinea pig.

The effects of levobunolol hydrochlorid (Bun) on the type L calcium channel currents (ICA) and delayed rectifier potassium channel currents (IK) in isolated ventricular myocytes of guinea pig were studied by using patch clamp whole cell recording techniques. The results were showed that: 1) Bun caused a dose dependent decrease in ICA and a dose-dependent increase in IK of the ventricular myocytes. The threshold concentrations of Bun for ICA and IK were 10(-8) mol/L and 10(-7) mol/L respectively. The maximum effective concentration of Bun for bot ICA and IK was 3x10(-5) mol/L, and half-maximal concentration was 3x10(-6) mol/L; 2) IK was blocked by 2x100(-6) mol/L tetraethylammonium (TEA). A concentration of 3x10(-6) mol/L Bun showed a decreasing effect on the ICA as revealed by the current-voltage relationship curve, i.e., Bun caused an elevation of the curve;3) When ICA was blocked by 2x10(-6) mol/L Isoptin (Verapamil), at a concentration of 3x10(-6) mol/L Bun showed an increasing effect on IK and the effect could be blocked by TEA. The above-mentioned results indicated that Bun had an inhibitory effect on ICA and a fascilitatory effect on IK. The results suggested that the molecular mechanisms of antihypertensive, heart rate slowing the beta -receptor blocking effects of Bun might be due to decrease of ICA and increase of IK.

Adrenergic beta-Antagonists↗
PubMed1997

Effect of asphyxia on urinary epidermal growth factor levels in newborns.

Urinary epidermal growth factor (EGF) excretion in normal newborn as well as neonates with asphyxia was investigated by using radioimmunoassay, and serum creatinine (Scr) levels determined at the same time. The results showed that in severe asphyxia group the ratio of urinary EGF to urinary creatinine (Cr) (EGF/Cr), an index reflecting EGF excretion, was decreased on the first day (P < 0.05) and reached the lowest level on the third day (P < 0.01). However, EGF/Cr values were decreased only on the third day in neonates with mild asphyxia (P < 0.05). On the seventh day, EGF/Cr values of neonates with asphyxia rose to normal. There were a negative correlation between urinary EGF/Cr and Scr. It is suggested that EGF may play a role in the repair of acute renal injury after asphyxia and the detection of urinary EGF concentration is useful in the judgment of severity of renal injury and in the evaluation of the recovery of renal tubule after injury.

Acute Kidney Injury↗
PubMed1997

A 54-kDa protein related to ras-guanine nucleotide release factor expressed in the rat exocrine pancreas.

The polymerase chain reaction was used to amplify a cDNA encoding the catalytic region of ras-guanine nucleotide release factor (ras-GRF1) from mouse embryonic stem cell mRNA. Antibodies directed against this protein were prepared and affinity purified. Western immunoblotting of rat tissue lysates revealed a 140-kDa protein in brain as expected but, in addition, a strongly immunoreactive 54-kDa protein, p54, was identified in pancreas. Expression of ras-GRF1 in pancreas was confirmed at the RNA level by reverse-transcriptase-coupled polymerase chain reaction analysis; p54 may therefore correspond to a form of ras-GRF1 or a closely related protein. The cellular and subcellular localization of p54 was investigated by enzyme-linked immunocytochemistry and immunogold electron microscopy. In the pancreas, p54 was expressed primarily in acinar cells, where it was localized along the basolateral and apicolateral plasma membranes. Indirect immunofluorescence microscopy of cultured acini further indicated that the plasma membrane localization of p54 was dependent on the maintenance of the acinar histotype and organized acinar structure. When primary acinar cells were permitted to dissipate into monolayer cultures devoid of zymogen granules, ras-GRF1 staining became cytosolic. Our results suggest that ras-GRF1 is involved in the structure and function of the pancreas.

Alternative Splicing↗
PubMed1997

Costimulation, tolerance and ignorance of cytolytic T lymphocytes in immune responses to tumor antigens.

Despite the fact that many tumors express MHC class I molecules presenting "foreign" peptide antigens, a vigorous tumor-destructing immune response is seldom detected. A possible explanation is that tumors cannot provide adequate costimulatory signals as provided by professional antigen presenting cells. CD28, upon interacting with B7, triggers costimulatory signals critical for the T-cell response. Transfection of tumor cells with B7 augments the immunogenicity of the tumor so that an anti-tumor immune response can be amplified. When B7-CD28 costimulation is provided CTL specific for otherwise silent epitopes can be activated. Therefore, unresponsiveness of T cells to many tumor antigens should be considered as ignorance rather than tolerance. Immunological ignorance may thus contribute to the failure of the immune system to respond against the tumor antigens.

Animals↗
PubMed1997

Folates and one-carbon metabolism in plants and fungi.

Folate-dependent pathways of one-carbon metabolism are essential for the synthesis of purines, formylmethionyl-tRNA, thymidylate, serine and methionine. These syntheses use a cellular source of one-carbon substituted, tetrahydrofolate polyglutamate derivatives which are the preferred substrates of most folate-dependent enzymes. In the last decade, there have been major advances in the folate biochemistry of animal, bacterial, fungal and plant systems. These have included the refinement of methods for folate isolation and characterization, basic work on key enzymes of folate biosynthesis and the detailed characterization of proteins that catalyze the generation and utilization of one-carbon substituted folates.

Folic Acid↗
PubMed1997

Associative learning.

This chapter reviews evidence demonstrating the essential role of the cerebellum and its associated circuitry in the learning and memory of classical conditioning of discrete behavioral responses (e.g., eyeblink, limb flexion, head turn). It now seems conclusive that the memory traces for this basic category of associative learning are formed and stored in the cerebellum. Lesion, neuronal recording, electrical microstimulation, and anatomical procedures have been used to identify the essential conditioned stimulus (CS) circuit, including the pontine mossy fiber projections to the cerebellum; the essential unconditioned stimulus (US) reinforcing or teaching circuit, including neurons in the inferior olive (dorsal accessory olive) projecting to the cerebellum as climbing fibers; and the essential conditioned response (CR) circuit, including the interpositus nucleus, its projection via the superior cerebellar peduncle to the magnocellular red nucleus, and rubral projections to premotor and motor nuclei. Each major component of the eyeblink CR circuit was reversibly inactivated both in trained animals and over the course of training. In all cases in trained animals, inactivation abolished the CR (and the UR as well when motor nuclei were inactivated). When animals were trained during inactivation (and not exhibiting CRs) and then tested without inactivation, animals with inactivation of the motor nuclei, red nucleus, and superior peduncle had fully learned, whereas animals with inactivation of a very localized region of the cerebellum (anterior interpositus and overlying cortex) had not learned at all. Consequently, the memory traces are formed and stored in the cerebellum. Several alternative possibilities are considered and ruled out. Both the cerebellar cortex and the interpositus nucleus are involved in the memory storage process, suggesting that a phenomenon-like long-term depression (LTD) is involved in the cerebellar cortex and long-term potentiation (LTP) is involved in the interpositus. The experimental findings reviewed in this chapter provide perhaps the first conclusive evidence for the localization of a basic form of memory storage to a particular brain region, namely the cerebellum, and indicate that the cerebellum is indeed a cognitive machine.

Animals↗
PubMed1997

Decreased IgG-Fc gamma RII dissociation kinetics in the presence of a protein antigen.

Total internal reflection fluorescence microscopy has been used to examine the interaction of a mouse monoclonal IgG2b, in the absence and presence of its protein antigen, with mouse Fc gamma RII in substrate-supported planar membranes. Equilibrium association and kinetic dissociation constants were measured for the antibody S6-34.11, which is specific for bovine prothrombin fragment 1 (BF1). These measurements showed that BF1 induces a statistically significant decrease (30-40%) in the IgG-Fc gamma RII dissociation kinetics. A corresponding increase in the equilibrium association constant was not observed, perhaps because the statistical accuracy of the equilibrium measurements is lower than that for the kinetic measurements. The consequences of these results for understanding the mechanism by which macrophages recognize and ingest opsonized targets are discussed.

Animals↗
PubMed1997

Influence of ablated tissue on the formation of high-intensity focused ultrasound lesions.

In order to ablate tumours using high-intensity focused ultrasound (HIFU) it is necessary to irradiate the tumour with a confluent array of single ultrasound exposures. We have identified a phenomenon that we term lesion-to-lesion interaction, which occurs when the spatial separation of individual exposures is such that an existing lesions appears to affect the formation of a subsequent lesion. This article investigates the implications of this phenomenon for strategies to ablate large tissue volumes in the treatment of hepatic metastases. Experiments on pig and rat livers have been carried out using a focused ultrasound system with a frequency of 1.7 MHz, an in situ spatially averaged focal intensity (ISAL) of 133-658 W cm-2 (ISP of 239-1185 W cm-2) and an exposure duration of 5-15 s. The results show that there is interaction between lesions that spatial exposure separations that depend on the intensities and exposure durations used. As a result, either subsequent lesions form closer to the ultrasound source (if the focal peak of the ultrasound beam is placed deep inside the liver tissue) or their length is reduced (if the focal peak is near the liver surface). An explanation is suggested for this effect and a strategy for its avoidance during in vivo HIFU treatment is discussed.

Animals↗
PubMed1997

Steady state EP is not responsible for hearing loss in adult chickens following acoustic trauma.

The steady state DC endocochlear potential (EP) in young chicks shows a large decrease after acoustic overstimulation followed by a rapid recovery that parallels the recovery of threshold (Poje et al., Hear. Res. 82 (1995) 197-204). These results raise a question as to whether or not the EP could account for the hearing loss and make a significant contribution to the recovery of the threshold. In contrast to results in young chicks, we show that acoustic overstimulation, which causes extensive hair cell damage, does not cause a decrease in the steady state EP in adult chickens. However, there is a significant reduction in the negative EP seen during anoxia which persists even after 4 weeks of recovery. Thus, our results indicate that the steady state EP cannot account for the hearing loss observed in adult chickens.

Acoustic Stimulation↗