Search PubMed⌕ Search

Biomedical subjects

D Chen

Publications and source records attributed to D Chen.

At least 487 records · Page 27Linked to original sources
PubMed1997

Expression of the chromogranin A-derived peptides pancreastatin and WE14 in rat stomach ECL cells.

The ECL cells constitute the predominant endocrine cell population in the mucosa of the acid-secreting part of the stomach (fundus). They are rich in chromogranin A (CGA), histamine and histidine decarboxylase (HDC). They secrete CGA-derived peptides and histamine in response to gastrin. The objective of this investigation was to examine the expression of pancreastatin (rat CGA266-314) and WE14 (rat CGA343-356) in rat stomach ECL cells. The distribution and cellular localisation of pancreastatin- and WE14-like immunoreactivities (LI) were analysed by radioimmunoassay and immunohistochemistry with antibodies against pancreastatin, WE14 and HDC. The effect of food deprivation on circulating pancreastatin-LI was examined in intact rats and after gastrectomy or fundectomy. Rats received gastrin-17 (5 nmol/kg/h) by continuous intravenous infusion or omeprazole (400 micromol/kg) once daily by the oral route, to induce hypergastrinemia. CGA-derived peptides in the ECL cells were characterised by gel permeation chromatography. The expression of CGA mRNA was examined by Northern blot analysis. Among all of the endocrine cells in the body, the ECL cell population was the richest in pancreastatin-LI, containing 20-25% of the total body content. Food deprivation and/or surgical removal of the ECL cells lowered the level of pancreastatin-LI in serum by about 80%. Activation of the ECL cells by gastrin infusion or omeprazole treatment raised the serum level of pancreastatin-LI, lowered the concentrations of pancreastatin- and WE14-LI in the ECL cells and increased the CGA mRNA concentration. Chromatographic analysis of the various CGA immunoreactive components in the ECL cells of normal and hypergastrinemic rats suggested that these cells respond to gastrin with a preferential release of the low-molecular-mass forms.

Animals↗
PubMed1997

Regulation of platelet plasma membrane Ca2+-ATPase by cAMP-dependent and tyrosine phosphorylation.

As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60(src) resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after 32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60(src). These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.

Blood Platelets↗
PubMed1997

Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.

Adenoviridae↗
PubMed1997

Pituitary adenylate cyclase-activating peptide (PACAP) in the retinohypothalamic tract: a potential daytime regulator of the biological clock.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing via a glutamatergic pathway at night. Here we report a new signaling pathway by which the RHT may regulate circadian timing in the daytime as well. We used dual immunocytochemistry for pituitary adenylate cyclase-activating peptide (PACAP) and the in vivo tracer cholera toxin subunit B and observed intense PACAP-immunoreactivity (PACAP-IR) in retinal afferents in the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR in the SCN as well as in the IGL was nearly lost after bilateral eye enucleation. PACAP afferents originated from small ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced (3.5 +/- 0.4 hr) by application of 1 x 10(-6) M PACAP-38 during the subjective day [circadian time (CT)-6] but not at night (CT14 and CT19). The phase-shifting effect is channeled to the clock via a PACAP-R1 receptor, because mRNA from this receptor was demonstrated in the ventral SCN by in situ hybridization. Furthermore, vasoactive intestinal peptide was nearly 1000-fold less potent in stimulating a phase advance at CT6. The signaling mechanism was through a cAMP-dependent pathway, which could be blocked by a specific cAMP antagonist, Rp-cAMPS. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT may adjust the biological clock by a PACAP/cAMP-dependent mechanism during the daytime.

Animals↗
PubMed1997

Stimulation of haemolytic activity of sea anemone cytolysins by 8-anilino-1-naphthalenesulphonate.

This study reports for the first time stimulation of protein activity by the hydrophobic probe, 8-anilino-1-naphthalenesulphonate (ANS). Magnificalysin (HMg) I and II and equinatoxin (EqTx) II and III are cytolysins isolated from the sea anemone Heteractis magnifica and Actinia equina, respectively. The haemolytic activity of these cytolysins could be stimulated by treatment with ANS. Their activation involved conformational changes following ANS treatment as shown by fluorescence spectra. ANS-induced conformational changes were reversible upon removal of ANS. ANS-stimulated activity of HMg I was inhibited by sphingomyelin and antiserum but not affected by bromosuccinimide (NBS) which oxidises tryptophan residues. However, toxin pre-treated with NBS could no longer be stimulated by addition of ANS. Energy transfer from tryptophan to ANS was observed by a fluorescence scan. Hence the tryptophan residues appear to be involved, at least partially, in ANS-binding. ANS-induced conformational change may be responsible for the activation of the cytolytic activity of these cytolysins.

Anilino Naphthalenesulfonates↗
PubMed1997

Insertion of the 34-kDa chloroplast protein import component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTP-binding capacity.

IAP34 is a 34-kDa component of the outer membrane complex that mediates the initial stages of protein import into chloroplasts (Seedorf, M., Waegemann, K., and Soll, J. (1995) Plant J. 7, 401-411; Kessler, F., Blobel, G., Patel, H. A., and Schnell, D. J. (1994) Science 266, 1035-1039). We have investigated the targeting and insertion of IAP34 at the outer envelope membrane. The analyses of IAP34 deletion mutants and hybrid proteins (consisting of regions of IAP34 fused to the soluble IgG-binding domain of staphylococcal protein A) suggest that the transmembrane domain and C-terminal tail of IAP34 contain information essential but not sufficient for targeting to the outer membrane. Treatment of chloroplasts with exogenous proteases does not affect IAP34 insertion, indicating that targeting does not require surface-exposed receptors at the envelope. GTP or GDP is required for maximal integration of IAP34 into the outer membrane. The GTP/GDP requirement is attributed to the intrinsic GTP binding activity of IAP34 because GTP/GDP binding-deficient mutants are defective in outer membrane insertion. On the basis of these observations, we propose that IAP34 is targeted to the chloroplast by a C-terminal signal and efficiently integrated into the outer membrane by conformation-induced insertion upon GTP/GDP binding.

Amino Acid Sequence↗
PubMed1997

A high-density screening format for encoded combinatorial libraries: assay miniaturization and its application to enzymatic reactions.

A novel, miniaturized high-throughput screening format is described for assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, utilizes the photolytic cleavage of individual compounds into a high-density well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 microl. As a model study, an encoded dipeptide library (324 members) acylated with isobutyl succinate was assayed using this format to search for potential inhibitors of matrilysin, a member of the matrix metalloproteinase superfamily. In situ release of compounds from solid support was accomplished by photochemical cleavage after beads and enzyme were distributed to the wells. After the addition of a fluorogenic substrate to the array, the extent of enzyme inhibition and identification of active compounds was quantitated by imaging of the fluorescence emission upon uv irradiation. The structure-activity relationship data generated from the identified inhibitors in this study corroborate previous findings, thus validating the utility of this approach as a means of high-throughput screening of bead-based libraries.

Acylation↗
PubMed1997

Evidence that rat stomach ECL cells represent the main source of circulating pancreastatin.

Recently, we showed that the ECL cells in the oxyntic mucosa of the rat stomach are an important source of circulating pancreastatin, a fragment of chromogranin A. The present study examined how much the ECL cells contribute to the circulating levels of pancreastatin during omeprazole-evoked hypergastrinemia. Rats received omeprazole (400 mumol kg-1 day-1) by the oral route for 3 weeks. Two weeks after the start of the treatment, the rats were subjected to a sham operation or fundectomy. The concentrations of gastrin and pancreastatin in serum were monitored before and after the operations. The ECL cells were visualized by pancreastatin immunostaining and their number was determined. The activity of oxyntic mucosal histidine decarboxylase (HDC) was measured before and after 2 weeks of omeprazole treatment. Omeprazole-induced hypergastrinemia resulted in elevated serum pancreastatin and increased oxyntic mucosal HDC activity. Pancreastatin-immunoreactive cells were equally numerous before and after 2 weeks of omeprazole treatment. After surgical removal of the ECL cells by fundectomy, the serum gastrin concentration remained high whereas the serum pancreastatin concentration decreased by 90%. We conclude that the ECL cells in omeprazole-treated rats are responsible for 90% of circulating pancreastatin.

Animals↗
PubMed1997

Effects of fasting on the expression of gastrin, cholecystokinin, and somatostatin genes and of various housekeeping genes in the pancreas and upper digestive tract of rats.

It is generally accepted that while the gene expression of many gut hormones in the pancreas and upper digestive tract is influenced by the prandial state, the expression of house-keeping genes (used as internal standards) is stable. We have analysed how food deprivation affects the messenger (m) RNA expression of gastrin, cholecystokinin (CCK), and somatostatin and of house-keeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin and 18S ribosomal (r) RNA. RNA, isolated from oxyntic, antral and duodenal mucosa and pancreas, was subjected to Northern blot analysis using complementary RNA probes. Compared to fed rats, food-deprived rats exhibited reduced mRNA expression of gastrin (antrum), somatostatin (antrum), CCK (duodenum), GAPDH (all tissues studied) and beta-actin (all tissues studied) and unchanged 18S rRNA expression. We conclude that the assessment of gut hormone mRNA expression may be greatly influenced by the choice of internal standard and that 18S rRNA is superior.

Actins↗
PubMed1997

Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1.

Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation. To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, we screened an adult human muscle cDNA library using the yeast two-hybrid cloning system. We report the isolation and characterization of a novel protein termed YAF2 (YY1- associated factor 2) that interacts with YY1. The YAF2 cDNA encodes a 180 amino acid basic protein (pI 10.5) containing a single N-terminal C2-X10-C2 zinc finger. Lysine clusters are present that may function as a nuclear localization signal. Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction. In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells. YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1. Co-transfection of YY1 reverses the stimulatory effect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium- activated protease m-calpain. The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development.

Actins↗
PubMed1997

RAB GTPases expressed in human melanoma cells.

The expression of small GTP-binding protein genes of the ras superfamily was examined in pigmented human melanoma cells by a PCR-based strategy. Twenty six different partial cDNA sequences were isolated, including 17 members of the rab subfamily, of which 9 represented novel genes. Some rabs expressed in melanoma cells overlapped with those of platelets: this should prove relevant to the investigation of murine and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.

Amino Acid Sequence↗
PubMed1997

Bone morphogenetic protein 2 transcripts in rapidly developing deer antler tissue contain an extended 5' non-coding region arising from a distal promoter.

To understand the regulation of the BMP-2 gene expression, we recently isolated the BMP-2 gene from a mouse genomic library and characterized the exon-intron structure and promoter. RNase protection assay using poly (A)+ RNA of mouse osteoblasts demonstrates that two regions in BMP-2 gene are protected by antisense mouse BMP-2 RNA probes. These results demonstrate that BMP-2 gene utilizes two alternative promoters, a distal and a proximal promoter. In the present study we demonstrate that BMP-2 mRNA from rapidly growing deer antler tissue has an extended 5' non-coding region compared with the human and rat BMP-2 mRNA. The extended 5' non-coding region in the deer mRNA represents transcripts from the upstream distal promoter. This is the first evidence of a natural BMP-2 mRNA from a bone-forming tissue that most likely initiated from the distal transcription start site.

Alternative Splicing↗
PubMed1997

Time and frequency domain analysis of acoustic signals from a human muscle.

We wished to determine whether evoked force output from a human muscle could be inferred from the amplitude and the frequency of evoked muscular sounds. Sounds from adductor pollicis were recorded with a microphone and compared to evoked force changes produced by varying the intensity of nerve stimulation or of voluntary effort as well as by fatigue and twitch potentiation. In all instances, high correlations were obtained between evoked acoustic signals amplitude and evoked twitch force changes. Instantaneous time-frequency distributions of evoked acoustic signals also showed a high degree of cross correlation with evoked instantaneous twitch force, but no relationship was found for the group between the maximal frequency of sound and force changes. We conclude that evoked force changes from human muscles cannot be reliably inferred from frequency domain analysis of evoked sound signals and that, for this purpose, analysis of sound signals in time domain can be employed, an analysis potentially useful for muscles whose force output cannot be directly measured.

Adult↗
PubMed1997

Molecular cloning and characterization of rab27a and rab27b, novel human rab proteins shared by melanocytes and platelets.

Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes, Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.

Amino Acid Sequence↗
PubMed1997

A fluoroquinolone antibiotic with a methoxy group at the 8 position yields reduced generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine after ultraviolet-A irradiation.

We have previously reported that two fluoroquinolone antibiotics gave rise to 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA of cells concurrently exposed to UV-A and that this correlated with clinical phototoxicity. To determine the structural basis for generation of oxidative damage, the ability of two synthetic fluoroquinolone candidate antibiotics, Bayer 12-8039 (12-8039) and Bayer Y3118 (Y3118), to give rise to 8-oxo-dG in cultured liver epithelial cells was compared. 12-8039 contains a methoxy group at the 8 position of the quinolone nucleus, whereas Y3118 contains a chlorine group at the same position. Y3118 produced dose-dependent increases in 8-oxo-dG formation in cultured cells after UVA irradiation, whereas the 8-OCH3-substituted 12-8039 produced no increase. Also, after exposure to 20 J/cm2 UVA, UV spectral scans of both compounds revealed that Y3118 underwent photodegradation whereas 12-8039 was stable. These results demonstrate that the presence of an 8-OCH3 group on the quinolone nucleus is important for the reduction of photogeneration of oxidative DNA damage and photodegradation in the presence of UVA irradiation. From this, we suggest that 12-8039 has little phototoxic potential.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Time-frequency analysis of the muscle sound of the human diaphragm.

The time-frequency characteristics of muscular sounds (phonomyogram) produced by the contraction of the human diaphragm under various contractile states is evaluated with the cone-kernel distribution. The results show that the instantaneous frequency of the phonomyogram of the diaphragm has a high cross-correlation (an average of 0.91 +/- 0.06) with the transdiaphragmatic pressure, with a delay varying between 25 and 35 ms. The instantaneous frequency response of the phonomyogram of the diaphragm shows a behaviour similar to that of the frog muscle; it rapidly rises and then fades out. However, the maximum of the instantaneous frequency of the phonomyogram of the diaphragm is not proportional to the maximum of the transdiaphragmatic pressure. This analysis also demonstrates the usefulness of the cone-kernel distribution for studying frequency-modulated signals like the muscular sound signals.

Diaphragm↗
PubMed1997

Time-frequency analysis of the first heart sound: Part 3: Application to dogs with varying cardiac contractility and to patients with mitral mechanical prosthetic heart valves.

The cone-kernel distribution (CKD) is first applied to the analysis of the intracardiac and the thoracic first heart sound (S1) of dogs in various cardiac contractile states, and secondly to the S1 of patients with mitral mechanical prosthetic heart valves. The CKD of native S1 in dogs shows that the dominant components of S1 are generally concentrated in a band at around 50 Hz with a horizontal flat or a semi-lunar shape, independently of the myocardial contractile state. There is no significant systematic rising frequency component. The instantaneous frequency of S1 shows a good cross-correlation with the time derivative of the left ventricular pressure (dP/dt), but the maximum frequency is not proportional to the maximum of dP/dt. The CKD of S1 in patients with mitral mechanical prosthetic heart valves showed a pulse-like component with a high-frequency bandwidth, which is distinct from the low constant-frequency components of S1 produced by native heart valves.

Animals↗