[Evaluation and treatment of the mentally ill with criminal records: a Calgary model].
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Alberta↗
Biomedical subjects
L Y Xie
Publications and source records attributed to L Y Xie.
MaRX: an approach to genetics in mammalian cells.
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Animals↗
Myc activates telomerase.
Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Here, we show that Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation.
Cell Division↗
Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone (PTH)/PTH-related peptide receptor.
The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.
Amino Acid Sequence↗
Downregulation of the PTH/PTHrP receptor by vitamin D3 in the osteoblast-like ROS 17/2.8 cells.
Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the parathyroid hormone (PTH)/PTH-related peptide (rP) receptor protein and mRNA in ROS 17/2.8 cells were studied. Treatment of ROS 17/2.8 cells with 1,25(OH)2D3 caused time- and dose-dependent suppression of PTH/PTHrP receptor number and immunoreactivity. The effects required more than 24 h incubation with 1,25(OH)2D3 and were maximal by 72 h. The cells did not recover their PTH/PTHrP receptors even after 4 days of treatment with control medium. Treatment with low concentrations of 1,25(OH)2D3 (0.1 M) dramatically decreased the PTH/PTHrP receptor mRNA levels, which were maximal after 24 h of incubation. The half-life of the PTH/PTHrP receptor transcript, 6-8 h, was similar in control and 1,25(OH)2D3-treated cells, suggesting that 1,25(OH)2D3 acts in controlling transcription of the PTH/PTHrP receptor gene but does not change the degradation rate of the PTH/PTHrP receptor transcripts. These data indicate that 1,25(OH)2D3 has a potent inhibitory effect on the expression of the PTH/PTHrP receptor protein and mRNA in ROS 17/2.8 cells.
Amino Acid Sequence↗
Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.
The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF.
Animals↗
The alternatively spliced type II corticotropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein(s).
Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5 fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RI was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 +/- 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 +/- 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-gamma-S inhibited hCRF binding to CRF-RI and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-gamma-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.
Alternative Splicing↗
Truncation of the carboxyl-terminal region of the rat parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylyl cyclase but not phospholipase C.
The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.
Adenylate Cyclase Toxin↗
Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor.
CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Down-regulation of parathyroid (PTH)/PTH-related peptide receptor immunoreactivity and PTH binding in opossum kidney cells by PTH and dexamethasone.
Recent data have shown that PTH down-regulation of its receptor on opossum kidney (OK) cells is not associated with any change in the steady state level of the PTH/PTH-related peptide (PTHrP) receptor messenger RNA. For analysis of down-regulation of the PTH/PTHrP receptor in OK cells, the present work uses a specific receptor anti-serum, SR-2, that is useful for detection and quantification of PTH/PTHrP receptor immunoreactivity on intact cells bearing the opossum PTH/PTHrP receptor. SR-2 specifically binds to COS-7 cells transiently expressing the opossum PTH/PTHrP receptor complementary DNA (OK-O), to LLCPK1 cells stably expressing the recombinant opossum PTH/PTHrP receptor (AOK cells), and to OK cells expressing endogenous PTH/PTHrP receptors, but not to mock-transfected COS-7 cells or untransfected LLCPK1 cells. SR-2 binding was also linearly correlated with PTH binding in COS-7 cells transfected with different amounts of OK-O plasmid DNA. Treatment with PTH (100 nM) for 4 and 6 h did not significantly down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased to 51% and 49% of control, respectively, and PTH-stimulated cAMP accumulation was decreased to 27% and 28% of control, respectively. Treatment with PTH (100 nM) for 24 and 48 h significantly decreased PTH binding to 51% and 60% of control and decreased PTH/PTHrP receptor immunoreactivity to 68% and 58% of control, respectively. Incubation of OK cells with 0.1 nM to 1 microM PTH for 4 h did not down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased dramatically. Scatchard blot analysis revealed that the binding affinity was decreased by 7-fold in OK cells treated with PTH for 4 h without change in receptor number. Conversely, treatment of OK cells with PTH for 24 h resulted in a parallel decrease in both receptor number and receptor immunoreactivity without any change in receptor binding affinity. Treatment of OK cells with dexamethasone (0.1 nM to 1 microM) had no effect on PTH binding or PTH/PTHrP receptor immunoreactivity. Incubation of OK cells with both dexamethasone (1 microM) and PTH (0.1 nM to 1 microM), however, caused a significantly greater down-regulation of both PTH binding and PTH/PTHrP receptor immunoreactivity than in cells treated with PTH alone. These data indicate that during the first 4 h of exposure of OK cells to PTH, PTH/PTHrP receptors remain on the cell surface but have lowered affinity to bind the ligand and that dexamethasone potentiates the effect of PTH on PTH/PTHrP receptor down-regulation in OK cells.
Animals↗
Effect of porphyrins on the hematopoietic recovery of mice treated with gamma-radiation.
Porphyrins are a group of organic compounds involved in a wide spectrum of fundamental biological processes. Non-metallic, naturally occurring and synthetic porphyrin derivatives may produce cytotoxic effects in malignant or normal tissues exposed to visible light. Supra-clinical doses of the photosensitizing porphyrin, Photofrin are hematostimulatory when administered to normal and immunosuppressed inbred mice. To determine if a non-photosensitizing metalloporphyrin has similar hematostimulatory activity, we have synthesized iron (III) hematoporphyrin chloride (FeHp) and administered it to sub-lethally irradiated mice. FeHp (10 mg/kg) given 1 and 4 days or 1, 4 and 7 days following sub-lethal (7 Gy) whole body irradiation significantly increased spleen colony forming units of progenitor cells of the granulocyte-macrophage lineage (CFU-GM) 14 days post-irradiation, relative to irradiated controls. In addition, total splenocyte numbers were significantly increased 17 days post-irradiation in mice that had received FeHp 1 and 4 days post-irradiation. When FeHp was given 24 hours prior to irradiation and again 48 hours or 48 and 96 hours post-irradiation, significant increases in splenic CFU-GM and spleen cell numbers, relative to control mice, were observed 15 days post-irradiation. A non-metallic photosensitizing monomeric fraction of Photofrin, deuteroporphyrin IX, 2,4 (4,2) hydroxyethyl vinyl (HVD) was compared to Photofrin for its ability to influence the hematopoietic recovery of irradiated mice. Only Photofrin but not HVD given in 3 doses (10 mg/kg) 1, 4 and 7 days following irradiation (4.8 Gy) significantly enhanced the recovery of spleen cellularity and splenic CFU-GM. In addition, Photofrin significantly increased bone marrow CFU-GM 7 and 10 days following the sub-lethal dose of gamma-radiation. The mechanism by which certain porphyrins augment hematopoiesis in the mouse is unknown. However, the identification of FeHp as a non-photosensitizing monomeric porphyrin with hematostimulatory activity in vivo, indicates that further study of metalloporphyrins is warranted and may reveal their clinical potential within the context of therapeutically-induced immunosuppression.
Animals↗
Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide.
Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.
1-Methyl-3-isobutylxanthine↗
[The difference of erythropoietin concentration in various disease].
The serum concentration of erythropoietin in 79 cases with various blood diseases, uremia, chronic obstructive pulmonary disease etc was determined. At comparable degrees of anemia, patients with myelodysplastic syndrome and aplastic anemia had the highest levels of erythropoietin in our study. The high level of erythropoietin titer in patients with aplastic anemia should be taken as the nom for renal synthesis and release of this hormone. The erythropoietin level in patients with uremic anemia was lower than the level in patients with anemia of other causes but still higher than that of the normal controls. Patients suffering from polycystic kidney disease with or without uremia had a high level of erythropoietin due to local hypoxia of remnant kidney tissue resulting from the pressure of cystic formation. Different methods are used to determine the erythropoietin level, which varies with the stage and etiology of the diseases. There are other stimulating or inhibitory factors of erythropoiesis when the assay is processed. Transfusion and administration of certain drugs also influence the growth of erythroid cells, thus the serum titers of erythropoietin differed markedly between patients at comparable hemoglobin concentration.
Adolescent↗
Treatment of 40 cases of paralysis of common peroneal nerve with acupuncture and moxibustion.
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Acupuncture Therapy↗