Biomedical subjects

H Wang

Publications and source records attributed to H Wang.

At least 325 records · Page 18Linked to original sources

PubMed2001

A micromorphometry-based concept for routine classification of sentinel lymph node metastases and its clinical relevance for patients with melanoma.

BACKGROUND: The sentinel lymph nodes (SLNs) as the primary targets for lymphatic metastases can be removed selectively by gamma probe-guided sentinel lymph nodectomy (SLNE) in nearly all patients with cutaneous melanoma. Correspondingly high standards in terms of specificity, sensitivity, and microstaging are required for the evaluation of SLNs. METHODS: Since 1995, the authors have performed SLNE in 389 lymph node regions (LNRs) on 342 patients with melanoma. The harvested 636 SLNs and a further 1394 nonsentinel lymph nodes (non-SLNs) were evaluated by standardized, semiserial section histology, including immunohistochemistry. For each LNR, this technique permitted routine S classification using two simple morphometric parameters: the number of tumor-involved, 1-mm slices of the SLNs (n) and the centripetal depth of metastatic cell invasion (d). S1 was defined by 1 < or = n < or = 2 and d < or = 1 mm, equivalent to localized peripheral tumor cell deposits; S2 was defined by n > 2 and d < or = 1 mm, indicating more extended peripheral metastases; S3 was defined by d > 1 mm in SNLs with deeper metastatic infiltration; and S0 meant no detectable tumor cells (n = 0). RESULTS: The authors diagnosed 325 SLNs as S0, 24 SLNs as S1, 22 SLNs as S2, and 18 SLNs as S3. The occurrence of at least one melanoma-positive non-SLN subsequent regional completion lymph node dissection (RCLND) significantly increased from 0 of 12 in S1 SLNs to 2 of 13 in S2 SLNs and 9 of 15 in S3 SLNs (P = 0.001; chi-square test). Like the T classification of the primary melanoma, the S classification proved to be a highly significant predictor for distant metastasis (P < 0.001). It turned out to be an independent factor of influence on distant metastasis and survival in multivariate Cox analyses, which included tumor thickness, primary tumor site, patient gender, and patient age as covariates. CONCLUSIONS: The data presented recommend the S-staging concept as a promising option to fill a gap between the T and conventional N component of the pTNM classification. If its predictive capacity can be confirmed in multicenter studies, then the S classification may become the decisive criterion for or against RCLND, and a combined T plus S staging system will help to improve prognostically relevant stratification of melanoma patients in adjuvant therapy trials.

Adult↗

PubMed2001

Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor.

Acetylation of core histone tails plays a fundamental role in transcription regulation. In addition to acetylation, other posttranslational modifications, such as phosphorylation and methylation, occur in core histone tails. Here, we report the purification, molecular identification, and functional characterization of a histone H4-specific methyltransferase PRMT1, a protein arginine methyltransferase. PRMT1 specifically methylates arginine 3 (Arg 3) of H4 in vitro and in vivo. Methylation of Arg 3 by PRMT1 facilitates subsequent acetylation of H4 tails by p300. However, acetylation of H4 inhibits its methylation by PRMT1. Most important, a mutation in the S-adenosyl-l-methionine-binding site of PRMT1 substantially crippled its nuclear receptor coactivator activity. Our finding reveals Arg 3 of H4 as a novel methylation site by PRMT1 and indicates that Arg 3 methylation plays an important role in transcriptional regulation.

Acetylation↗

PubMed2001

Neurokinin-1 receptor-immunoreactive neurons of the ventral respiratory group in the rat.

The rostral end of the ventral respiratory group (VRG) contains neurons that are intensely neurokinin-1 receptor (NK1R) immunoreactive (ir). It has been theorized that some of these cells might be critical to respiratory rhythmogenesis (Gray et al. [1999] Science 286:1566-1568). In the present study we determined what major transmitter these NK1R-ir cells make and whether they are bulbospinal or propriomedullary. NK1R-ir neurons were found in the VRG between Bregma levels -11.7 and -13.6 mm. The highest concentration was found between Bregma -12.3 and -13.0 mm. This region overlaps with the pre-Bötzinger complex (pre-BötC) as it was found to contain many pre-inspiratory neurons, few E2-expiratory neurons, and no I-incremental neurons. VRG NK1R-ir neurons contain neither tyrosine hydroxylase (TH) nor choline acetyl-transferase (ChAT) immunoreactivity, although dual-labeled neurons were found elsewhere within the rostral medulla. GAD67 mRNA was commonly detected in the ventrolateral medulla (VLM) but rarely in the NK1R-ir neurons of the pre-BötC region (6 % of somatic profiles). GlyT2 mRNA was commonly found in the pre-BötC region but rarely within NK1R-ir neurons (1.3 %). Up to 40% of VRG NK1R-ir neurons were retrogradely labeled by Fluoro-Gold (FG) injected in the contralateral pre-BötC region. Some NK1R-ir VRG neurons located caudal to Bregma -12.6 mm were retrogradely labeled by FG injected in the spinal cord (C4-C5, T2-T4). In sum, NK1R immunoreactivity is present in many types of ventral medullary neurons. Within the VRG proper, NK1R-ir neurons are concentrated in an area that overlaps with the pre-BötC. Within this limited region of the VRG, NK1R-ir neurons are neither cholinergic nor catecholaminergic, and very few are gamma-aminobutyric acid (GABA)ergic or glycinergic. The data suggest that most NK1R-ir neurons of the pre-BötC region are excitatory. Furthermore, the more rostral NK1R-ir cells are propriomedullary, whereas some of the caudal ones project to the spinal cord.

Acetylcholine↗

PubMed2001

Absolute configuration and conformational stability of (+)-2,5-dimethylthiolane and (-)-2,5-dimethylsulfolane.

Enantiopure (+)-2,5-dimethylthiolane and (-)-2,5-dimethylsulfolane were prepared using literature procedures and investigated using vibrational circular dichroism (VCD). Experimental absorption and VCD spectra of (+)-2,5-dimethylthiolane and (-)-2,5-dimethylsulfolane in CCl(4) solution in the 2000-900 cm(-)(1) region were compared with the ab initio predictions of absorption and VCD spectra obtained with density functional theory using the B3LYP/6-311G(2d, 2p) basis set for different conformers of (2R,5R)-2,5-dimethylthiolane and (2R,5R)-2,5-dimethylsulfolane. This comparison indicates that (+)-2,5-dimethylthiolane is of the (2R,5R)-configuration and has two predominant conformations in CCl(4) solution. In addition, (-)-2,5-dimethylsulfolane is of (2R,5R)-configuration and has only one predominant conformation. The stereochemical assignment is in agreement with literature.

Journal Article↗

PubMed2001

Anionic photofragmentation of CO: a selective probe of core-level resonances.

Anion-yield spectroscopy using x rays is shown to be a selective probe of molecular core-level processes, providing unique experimental verification of shape resonances. For CO, partial anion and cation yields are presented for photon energies near the C K edge. The O- yield exhibits features above threshold related only to doubly excited states, in contrast to cation yields which also exhibit pronounced structure due to the well-known sigma* shape resonance. Because the shape resonance is completely suppressed for O-, anion spectroscopy thus constitutes a highly selective probe, yielding information unobtainable with absorption or electron spectroscopy.

Journal Article↗

PubMed2001

Different processes are involved in human brain for shape and face comparisons.

Fifteen subjects participated in a matching task of visual stimuli. Two sequentially presented stimuli in a pair were the same shape (shape match), different shapes (shape mismatch), same human face (face match) or different faces (face mismatch). All four kinds of stimulus pairs were of equal probability. The shape mismatch pairs elicited a negative event-related potential component N270 (Peak latency: 262.1+/-16.5 ms, P4) after the second stimulus onset, while the face mismatch evoked N270 with longer peak latency (301.2+/-19.8 ms, P4) and N450. There exists a specific system for mismatch processing of a complex stimulus (face) in the human brain.

Cerebral Cortex↗

PubMed2001

[Mycophenolate mofetil in the treatment of primary nephrotic syndrome].

OBJECTIVE: To investigate the efficacy and safety of mycophenolate mofetil (MMF) on treating refractory primary nephrotic syndrome. METHODS: Forty-one patients with refractory nephrotic syndrome confirmed by renal biopsy, 19 with minor lesion (minimal lesion nephropathy and mesangial proliferative glomerulonephritis), 18 with membranous nephropathy (MN), 3 with focal segmental glomerulosclerosis (FSGS), and one with mesangioproliferative glomerulonephritis (MPGN), were treated by MMF combined with prednisone. The initial dosage of MMF was 1.0 - 2.0 g/d for three months and then the dosage was tapered gradually. The duration of MMF treatment was at least six months. Prednisone at the dose of 20 - 60 g/d was used at the beginning of the combined treatment and then the dosage was tapered gradually. Follow-up interview was conducted regularly. Four patients were rebiopsyed by the end of observation. RESULTS: The combined treatment of MMF/prednisone decreased the urine protein and elevated the serum albumin significantly among patients with minor lesion and MN (P < 0.001). All patients with minor lesion achieved clinical remission. Eleven of the nineteen cases responded within four weeks, and twelve of them obtained complete clinical remission. The dosage of prednisone could be tapered smoothly among the steroid dependent patients. Thirteen of the eighteen patients with MN achieved remission, however only six responded within four weeks and only three of them achieved complete clinical remission. During the treatment, four patients experienced transient increase of urine protein due to infection and recovered spontaneously without alteration of treatment. Side effects were tolerable except one case was withdrawn due to the decrease of hemoglobin. Renal function remained stable during the treatment. No obvious alteration could be found in renal biopsy by the end of treatment among four patients. CONCLUSION: MMF is an effective and safe immunosuppressive agent for refractory nephrotic syndrome.

Adolescent↗

PubMed2001

2'-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids.

We have synthesized two novel phosphoramidites with a ferrocenyl moiety at the 2'-ribose position linked through a butoxy linker. Using automated DNA/RNA synthesis techniques, oligonucleotides containing ferrocene at various positions were prepared and characterized by HPLC, MALDI-TOF mass spectrometry, and electrochemistry. Thermal stability studies of the ferrocene-modified DNA duplexes revealed that introduction of one or two ferrocenyl complexes does not result in an observed change of the T(m) values of the corresponding DNA duplexes when compared to the nonmodified hybrids. These data indicate that the introduction of a ferrocenyl group at the 2'-position of the ribose ring containing either a purine or pyrimidine base has no effect on the stability of the modified DNA. The electrochemical behavior of the ferrocene-containing DNA was examined by cyclic voltammetry. The modified 2'-ferrocene-oligonucleotides are electrochemically active and can be used as signaling probes for the electronic detection of nucleic acids on bioelectronic sensors.

Chromatography, High Pressure Liquid↗

PubMed2001

Effects of all-trans-retinoic acid and arsenic trioxide on the hemostatic disturbance associated with acute promyelocytic leukemia.

To study the in vivo effect of all-trans-retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) on the expression of tissue factor (TF) and the other hemostatic disturbance, a series of parameters were measured either in bone marrow blasts or plasma from acute promyelocytic leukemia (APL) patients. The plasma parameters were measured by ELISA or chromogenic studies. The TF transcription was assessed using reverse transcription-polymerase chain reaction (RT-PCR) technique. The results indicated that the blast cell procoagulant activity (PCA), TF antigen of APL cell lysate, as well as the transcription of APL TF mRNA elevated at diagnosis, were reduced after ATRA or As(2)O(3) therapy. The plasma level of P-selectin, TF, thrombin-antithrombin complex (TAT), soluble fibrinmonomer complex, thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), plasmin-antiplasmin complex, tissue plasminogen activator (t-PA) activity, urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and D-dimer (D-D) significantly increased. Fibrinogen (Fg), antigen level of protein C (PC), plasminogen (PLG) activity, alpha(2)-plasminogen inhibitor activity (alpha(2)-PI), and plasminogen activator inhibitor (PAI) activity were decreased at diagnosis. The protein C activity (PC:A) and protein S (PS) remained unchanged. All the parameters were restored to normal ranges after complete remission (CR) except elevation of TF and TAT in both groups, as well as PC:A, PS, and t-PA in the ATRA group. In conclusion, there existed activation of platelets and consumption of anticoagulants as well as activation of coagulation and fibrinolytic system before treatment. Both ATRA and As(2)O(3) therapy downregulated the expression of TF mRNA, decreased the PCA and TF level in APL cells, significantly inhibited coagulation activation, corrected secondary hyperfibrinolysis and the other hemostatic abnormalities, and thus greatly improved the bleeding symptom in early stage of the treatment.

Adolescent↗

PubMed2001

Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches: comparison with plasmalemmal mu-opioid receptors.

The activation of delta-opioid receptors (DORs) in the caudate-putamen nucleus (CPN) produces regionally distinct changes in motor functions, many of which are also influenced by opioids active at micro-opioid receptors (MORs). These actions most likely occur in MOR-enriched patch compartments in the CPN. To determine the functional sites for DOR activation and potential interactions involving MOR in these regions, immunoperoxidase and immunogold-silver labeling methods were applied reversibly for the ultrastructural localization of DOR and MOR in single rat brain sections containing patches of the CPN. DOR immunoreactivity was commonly seen within the cytoplasm of spiny and aspiny neurons, many of which also expressed MOR. In dendrites and spines, DOR labeling was preferentially localized to membranes of the smooth endoplasmic reticulum and spine apparatus, whereas MOR showed a prominent plasmalemmal distribution. DOR- and/or MOR-labeled spines received asymmetric, excitatory synapses, some of which showed notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids.

Animals↗

PubMed2001

Oxidative preconditioning and apoptosis in L-cells. Roles of protein kinase B and mitogen-activated protein kinases.

Oxidative stress can cause significant cell death by apoptosis. We performed studies in L-cells to explore whether prior exposure to oxidative stress ("oxidative preconditioning") can protect the cell against the apoptotic consequences of subsequent oxidative insults and to establish the mediators in the preconditioning signaling cascade. Cells were preconditioned with three 5-min exposures to H(2)O(2), followed by 10-h recovery and subsequent exposure to 600 microm H(2)O(2) for 10 h. A single 10-h exposure to H(2)O(2) induced substantial apoptotic cell death (approximately 90%), as determined by enzyme-linked immunosorbent assay, TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling), and Annexin V methods, but apoptosis was largely prevented in preconditioned cells. The degree of cytoprotection depended on the strength of preconditioning or H(2)O(2) concentration (20 approximately 600 microm). Transient increases in mitogen-activated protein kinase (MAPK), p38, and JNK/SAPK activities and sustained protein kinase B (Akt) activation, accompanied by drastically reduced caspase 3 activity, were seen after preconditioning. The expression levels of these kinases were unaltered. Inhibitors of p38 (SB203580) and phosphoinositide 3-kinase (PI3K, LY294002) pathways abolished the protection provided by preconditioning. We conclude that oxidative preconditioning protects cells against apoptosis and that this effect involves MAPK and PI3K/Akt pathways. This system may be important in regulating apoptotic cell death in development and disease states.

Animals↗

PubMed2001

[Infection of mutated mouse complement receptor type II by Epstein-Barr virus].

Site-directed mutagenesis method was used to introduce two desired mutations, which were confirmed by DNA sequencing, into mouse complement receptor Type II gene(MCR2). Then the constructed eukaryotic expression vectors containing wild type mouse CR2/1(wtMCR2/1), mutant type mouse CR2/1 (mtMCR2/1) and human CR2 (hCR2) cDNA were transferred into mouse SP2/0 cells by electroporation. After two-week screening by G418, the stably transfected clones were obtained. Several ways including PCR, RT-PCR, and immunohistochemistry were utilized to screen those clones with interesting genes integrated and expressed. Then Epstein-Barr virus(EBV) was used to infect these transfected cells and EBER-1 (EBV encoded RNAs) hybridization results showed that only hCR2 and mtMCR2 transfected SP2/0 cells could be infected by EBV, but positive rate of the former was much higher than the latter. This study sets groundwork for elucidating the mechanism by which EBV enters the cells and for establishing the animal model of EBV-related nasopharyngeal carcinoma (NPC).

Animals↗

PubMed2001

Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group.

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows biphasic kinetics with a fast (t(50): 5-30 min) component attributed to DNA-PK-dependent non-homologous endjoining (NHEJ) and a slow (t(50): 1-20 h), as of yet uncharacterized, component. To examine whether homologous recombination (HR) contributes to DNA DSB rejoining, a systematic genetic study was undertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR. We show that DT40 cells rejoin IR-induced DNA DSBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells with 1000-fold lower levels of HR. We also show that deletion of RAD51B, RAD52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD51. A significant reduction (to 37%) in the contribution of the fast component is observed in Ku70(-/-) DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs. A double mutant Ku70(-/-)/RAD54(-/-) shows similar half times to Ku70(-/-) cells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR. We propose that, in contrast to yeast, cells of vertebrates are 'hard-wired' in the utilization of NHEJ as the main pathway for rejoining of IR-induced DNA DSBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.

Animals↗

PubMed2001

[Expression and clinopathological significance of HCCA1 in hepatocellular carcinoma].

OBJECTIVE: To explore the correlation of a newly cloned HCCA1 gene expression with hepatocellular carcinoma (HCC). METHODS: HCCA1 mRNA and protein expression were detected by Northern blotting and by immunohistochemistry respectively. Its clinopathological significance was also analyzed. RESULTS: HCCA1 mRNA expression was found in 59 (90.8%) out of 65 patients. Of these, 54 (91.5%) showed significantly elevated expression of HCCA1 mRNA in HCC tissues. There were 9 (13.8%) patients who had low level of HCCA1 mRNA expression in paracarcinoma, and 56 (86.2%) cases showed no detectable signals. HCCA1 mRNA expression was closely related to lower tumor grading and portal vein invasion. It was noted that HCCA1 mRNA levels were higher in tumor grading III-IV group than in grading I-II group, higher in portal vein thrombus group than in non-thrombus group. Immunohistochemical staining confirmed that the HCCA1 protein was preferentially expressed in HCC tissues. Amongst 38 patients examined, 20 (52.6%) revealed positive staining with anti-HCCA1. CONCLUSION: HCCA1 is a novel HCC associated gene which may play a role in HCC invasion and metastasis.

Adult↗

PubMed2001

[The antagonistic effects of adrenomedullin on biological events governed by transforming growth factor beta in HK-2 cell line].

OBJECTIVE: To investigate the influence of adrenomedullin (AM) on the biological events governed by transforming growth factorbeta (TGF-beta) in human tubular epithelial cell line (HK-2). METHODS: Cell proliferation was determined by (3)H-TdR incorporation; ELISA method was used to detect the level of secreted fibronectin (FN); total collagen synthesis and secretion were reflected by (3)H-proline incorporation and the radioactivity of (3)H-hydroproline in the culture medium; the expression of FN mRNA, collagen IV mRNA and TIMP-1mRNA was determined by RT-PCR; plasma AM was measured by radioimmunoassay. RESULTS: (1) AM had no effect on cell growth inhibition of TGF-beta(1) (P > 0.05); (2) AM dose-dependently inhibited collagen synthesis and secretion stimulated by TGF-beta(1). As compared with those in TGF-beta group, collagen synthesis and secretion in AM (10(-8) mol/L group were inhibited by 8% (P > 0.05) and 30% (P < 0.05), respectively; and the collagen synthesis and secretion in AM (10(-7) mol/L) group were inhibited by 57% (P < 0.05) and 64% (P < 0.01), respectively in AM (10(-7) mol/L) group. Similarly, AM (10(-8) mol/L) inhibited the FN secretion (20 ng/microliter +/- 5 ng/microliter) stimulated by TGF-beta(1) (28 ng/microliter +/- 6 ng/microliter) (P < 0.05). RT-PCR showed that AM significantly down-regulated the expression of collagen IV, FN and TIMP-1 mRNA stimulated by TGF-beta(1). (3) The plasma AM in patients with mild tubulointerstitial lesion was 1. 2 times higher than that in normal controls. the plasma AM in patients with severe tubulointerstitial lesion was 2.2 times higher than that in normal controls. The plasma AM in patients with severe tubulointerstitial lesion was 50% higher than that in patients with mild tubulointerstitial lesion (P < 0.01). CONCLUSIONS: AM antagonizes the biological action of TGF-beta(1) on extracellular matrix accumulation; plasma AM increases in glomerulonephritis patients and correlates with the degree of tubulointerstitial lesion.

Adolescent↗

PubMed2001

A randomized efficacy and safety trial of oxandrolone in the treatment of Duchenne dystrophy.

BACKGROUND: A pilot study suggested that oxandrolone, an anabolic steroid, improved strength in boys with Duchenne dystrophy (DD) and indicated the need for a more definitive study. METHODS: A 6-month, randomized, double-blind, placebo-controlled study of oxandrolone in boys with an established diagnosis of DD, using the change from baseline to 6 months in the average muscle strength score (MMT) as the primary efficacy measure. RESULTS: The mean change from baseline for the oxandrolone group was +0.035 and that for the placebo group was -0.140. Although the oxandrolone group did not get worse and the placebo patients showed some deterioration in strength, the difference was not significant (p = 0.13). The average of the four quantitative muscle tests (QMT) showed a significant improvement in the oxandrolone-treated boys as compared with placebo. No adverse reactions attributable to oxandrolone were recorded. CONCLUSIONS: Although oxandrolone did not produce a significant change in the average manual muscle strength score as compared with placebo, the mean change in QMT was significant. Because oxandrolone is safe, accelerates linear growth, and may have some beneficial effect in slowing the progress of weakness, it may be useful before initiating corticosteroid therapy.

Anabolic Agents↗

PubMed2001

Agonist-induced mu opioid receptor phosphorylation and functional desensitization in rat thalamus.

By metabolically labeling tissue slices from striatum and thalamus with [32P]orthophosphoric acid and immunoprecipitating the receptor with mu receptor-specific antiserum, we found that the endogenous mu receptor in the brain tissue did undergo phosphorylation. The phosphorylation occurred at basal level (no drug treatment) and was enhanced with DAMGO-treatment. The enhancement of the phosphorylation was blocked by naloxone. Morphine stimulation also increased the phosphorylation, but the amount of enhancement was less than that caused by DAMGO-treatment. Mu receptor phosphorylation in the thalamus was much greater than the striatum, while no phosphorylation of the mu receptor in the cerebellum was detected, even with DAMGO treatment. The extent of mu receptor phosphorylation identified in the thalamus, striatum and cerebellum is consistent with the previous studies of mu receptor distribution. The time course and dose-response studies demonstrated that mu receptor phosphorylation was a rapid event, exhibited a positive dose-dependent response, and was similar to that observed in the cloned mu receptor in CHO cells. Furthermore, we correlated the change of mu receptor phosphorylation with the desensitization of the mu receptor function, specifically, inhibition of adenylyl cyclase activity in the thalamus of morphine-tolerant rats. We found that in the thalamus of rats chronically treated with morphine, the enhancement of mu receptor phosphorylation in basal and DAMGO-treated samples paralleled the desensitization of DAMGO-mediated inhibition of adenylyl cyclase. Our results suggest that mu receptor phosphorylation in vivo may play an important role in the modulation of mu receptor function following both acute exposure to morphine and during the development of morphine tolerance.

Adenylyl Cyclases↗

PubMed2001

The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.

The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.

A Kinase Anchor Proteins↗