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1.
Front Mol Biosci ; 9: 894207, 2022.
Article in English | MEDLINE | ID: mdl-35847976

ABSTRACT

Coronavirus disease 2019 (COVID 19) is a systemic infection that exerts a significant impact on cell metabolism. In this study we performed metabolomic profiling of 41 in vitro cultures of peripheral blood mononuclear cells (PBMC), 17 of which displayed IgG memory for spike-S1 antigen 60-90 days after infection. By using mass spectrometry analysis, a significant up-regulation of S-adenosyl-Homocysteine, Sarcosine and Arginine was found in leukocytes showing IgG memory. These metabolites are known to be involved in physiological recovery from viral infections and immune activities, and our findings might represent a novel and easy measure that could be of help in understanding SARS-Cov-2 effects on leukocytes.

2.
Support Care Cancer ; 27(4): 1255-1261, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30406916

ABSTRACT

PURPOSE: Hyponatremia and bone metastasis (BMs) are known as negative prognostic factors in patients affected by metastatic non-small cell lung cancer (NSCLC). Hyponatremia is associated with higher risk of osteoporosis and bone fracture, but no data are available about the relationship between hyponatremia and bone metastasis. This study aims to analyze the prognostic impact of hyponatremia in NSCLC patients with bone metastases. METHODS: We retrospectively collected data about advanced NSCLC patients. Survival curves were estimated using Kaplan-Meier method, and comparisons were made using chi-square test. RESULTS: Six hundred forty-seven patients were enrolled into the study. BMs were present in 264 patients (41%) at diagnosis, while hyponatremia appeared in 237 (37%) patients during the first-line treatment. Patients without BMs had a median overall survival (mOS) of 15.9 months (95% CI 14.1-17.9) versus 11.4 months (95% CI 9.4-13.4) for patients with BMs (p = 0.001). Eunatremic patients had a better outcome (mOS 16.3 months, 95% CI 14.6-18.0 vs 10.3 months, 95% I 7.6-12.8, p = 0.003). Considering the two variables, patients with BMs and hyponatremia had a mOS of 10.1 months (95% CI 4.3-15.9), patients with hyponatremia without BMs 11.9 months (95% CI 11.4-12.4), while mOS was 13.1 months (95% CI 12.0-14.2) for eunatremic patients with BMs versus 17.1 months (95% CI 15.2-19.1) in eunatremic patients without BMs (p = 0.0020). Hyponatremic patients developed metachronous BMs significantly earlier (3.73 vs 5.76 months, p = 0.0187). CONCLUSIONS: Our study showed that hyponatremia is an important prognostic factor and it should be necessarily considered to enhance the management of NSCLC patients with BMs.


Subject(s)
Bone Neoplasms/secondary , Carcinoma, Non-Small-Cell Lung/complications , Carcinoma, Non-Small-Cell Lung/pathology , Hyponatremia/diagnosis , Hyponatremia/etiology , Lung Neoplasms/complications , Lung Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/complications , Bone Neoplasms/diagnosis , Bone Neoplasms/therapy , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/therapy , Disease Progression , Female , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/therapy , Male , Middle Aged , Molecular Targeted Therapy , Neoadjuvant Therapy , Predictive Value of Tests , Prognosis , Retrospective Studies
3.
Support Care Cancer ; 23(10): 3095-101, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25739755

ABSTRACT

PURPOSE: Hyponatremia is the most common electrolyte disorder in hospitalized patients, and it might be an indicator of poor prognosis and might have negative effects on hospitalization length and quality of life in non-malignant as well as in malignant diseases. The aim of this study is to determine the impact of hyponatremia on the length and on the cost of hospitalization as well as on outcome in cancer patients. METHODS: The present study includes 105 consecutive cancer patients hospitalized at our institution from June 2013 to December 2013. Data regarding age, sex, staging, histology, chemotherapy, and serum sodium levels at admission, during hospitalization, and at discharge were recorded and statistically analyzed. Impact of hyponatremia on length and cost of hospitalization and on outcome was evaluated. RESULTS: A significant difference in overall survival since the date of admission was observed between eunatremic and hyponatremic patients (p = 0.0255). A statistically significant correlation was also found between the length of stay and the detection of hyponatremia. At multivariate analysis, hyponatremia at admission, severity of hyponatremia, and stage of disease resulted independent prognostic factors. Furthermore, a patient with moderate or severe hyponatremia cost, in rate terms, 128 and 299 % more than a normonatremic patient, respectively. CONCLUSIONS: The occurrence of hyponatremia at the admission or during the hospitalization may represent a significant factor influencing the outcome and the length of hospitalization. Acting effective and timely on the normalization of sodium levels might have a positive effect on prognosis in this setting of patients, as well as on the length of stay in hospital, thus potentially resulting in savings.


Subject(s)
Hyponatremia/blood , Neoplasms/complications , Neoplasms/economics , Adult , Aged , Aged, 80 and over , Female , Humans , Length of Stay , Male , Middle Aged , Multivariate Analysis , Neoplasms/blood , Quality of Life , Treatment Outcome
4.
Support Care Cancer ; 23(3): 621-6, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25142706

ABSTRACT

PURPOSE: Hyponatraemia is one of the most common tumour-related electrolyte disorders. Several clinical, histological and serum factors have been found to influence prognosis, but, to date, there are no studies focusing on the prognostic role of hyponatraemia in mesothelioma. The aim of this study was to assess the prognostic role of hyponatraemia in malignant pleural mesothelioma. METHODS: We analysed 62 consecutive patients with histologically or cytologically proven advanced malignant pleural mesothelioma undergoing chemotherapy at our institution between January 2003 and September 2013. RESULTS: All patients received a first-line pemetrexed-based chemotherapy. A second-line chemotherapy was administered to 29 patients. The onset of hyponatraemia (serum sodium <135 mEq/L) during the treatment was significantly related to a worsened median overall survival (7.93 vs 13.48 months; p = 0.0069). The occurrence of hyponatraemia during first-line chemotherapy (cutoff 135 and 130 mEq/L) was significantly associated to a shorter median progression-free survival (p = 0.0214). Results were also similar in the subgroup receiving a second-line treatment. At the multivariate analysis, including haemoglobin and sodium level at the beginning of first-line chemotherapy, age, gender, smoking habit, job exposure and performance status, only hyponatraemia was found to be an independent factor (p = 0.029). Hyponatraemia was also found to be a predictive factor for both first-line chemotherapy, being related to poorer response to pemetrexed-based chemotherapy (p = 0.047) and second-line chemotherapy (p = 0.044). CONCLUSION: Our results show that hyponatraemia might be considered a negative prognostic parameter in malignant pleural mesothelioma patients. To our knowledge, this is the first study to evaluate the association of hyponatraemia with the outcome of malignant pleural mesothelioma patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Hyponatremia/diagnosis , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mesothelioma/drug therapy , Mesothelioma/pathology , Pleural Neoplasms/drug therapy , Pleural Neoplasms/pathology , Aged , Aged, 80 and over , Carboplatin/administration & dosage , Carboplatin/adverse effects , Female , Glutamates/administration & dosage , Glutamates/adverse effects , Guanine/administration & dosage , Guanine/adverse effects , Guanine/analogs & derivatives , Humans , Hyponatremia/chemically induced , Hyponatremia/mortality , Lung Neoplasms/mortality , Male , Mesothelioma/mortality , Mesothelioma, Malignant , Middle Aged , Multivariate Analysis , Pemetrexed , Pleural Neoplasms/mortality , Prognosis , Survival Analysis
5.
Fish Shellfish Immunol ; 28(2): 303-11, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19925869

ABSTRACT

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.


Subject(s)
Bass/immunology , Bass/virology , Fish Diseases/immunology , Nodaviridae/immunology , RNA Virus Infections/veterinary , Animals , Antibodies, Viral/blood , Cell Line , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Fish Diseases/virology , Lymphocytes/cytology , Polymerase Chain Reaction , RNA Virus Infections/immunology
6.
Int J Sports Med ; 28(8): 662-6, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17455118

ABSTRACT

The aim of this study was to investigate the physiological load of free practices (F), qualifying sessions (Q), and official races (R) of motorcycling competitions. Twenty-six female riders (age: 30.8 +/- 6.1 yrs) participated in this study. Anthropometric and strength evaluations were performed. Subject's heart rate (HR) was continuously recorded during three periods (5-minute Pre, Ride, and 5-minute Post) of the F, Q, and R phases of competitions. Blood lactate concentrations (La) were determined at rest and after riding. Riders had 56.5 +/- 6.7 kg of body mass, 164 +/- 4 cm of height, 21.6 +/- 4.4 % of body fat, and a BMI of 20.9 +/- 2.3 kg . (m (2)) (-1). Significant handgrip differences were found between the preferred (307 +/- 32 n) and non-preferred (281 +/- 47 N) limbs. During riding, frequency of occurrence of HR >or= 90 % HR (max) was 67 % in F, 74 % in Q, and 83 % in R. La after ridings (4.8 +/- 2.6 mmol . l (-1)) was two-folds (p < 0.05) the basal values (2.2 +/- 0.5 mmol . l (-1)), independently from the competition phases. The present HR and La data show that female road race motorcycling imposes a very intensive physical strain to the riders who need to control their motorbikes while performing frequent accelerations and decelerations.


Subject(s)
Heart Rate/physiology , Lactic Acid/analysis , Motorcycles , Sports , Adult , Female , Humans , Italy , Lactic Acid/blood
7.
J Neurochem ; 79(5): 1047-58, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11739618

ABSTRACT

In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Delta425, Delta379, Delta351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys(351) and Asp(425) may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Delta351 is significantly increased in comparison with WT, Delta425 and Delta379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Delta351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys(351) may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Delta351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.


Subject(s)
Cytoplasm/metabolism , Dopamine/metabolism , Receptors, Dopamine D1/metabolism , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Primers , Dopamine Agonists/metabolism , GTP-Binding Proteins/metabolism , Humans , Immunohistochemistry , Molecular Sequence Data , Protein Conformation , Radioligand Assay , Rats , Receptors, Cell Surface/metabolism , Receptors, Dopamine D1/genetics
8.
Trends Endocrinol Metab ; 12(10): 453-60, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11701344

ABSTRACT

Studies of the serotonin (5-HT) receptors have illustrated several important concepts in G-protein-mediated signaling. These concepts include G-protein specificity and cellular specificity of signaling; mechanisms of transactivation; receptor states and constitutive receptor activity; and the structural basis of coupling. The 5-HT1 receptors couple via specific G(i)/G(o) proteins to inhibitory pathways [inhibition of adenylyl cyclase (AC) activity and regulation of ion channels], but also to stimulate phospholipase C, ACII, and the mitogen-activated protein kinase (MAPK) growth-signaling pathway. 5-HT1 receptors initiate novel endocytotic and Ca(2+)-dependent pathways to activate MAPK acutely, but can downregulate MAPK on chronic activation. These pathways are often mediated via distinct G(i)/G(o)-protein subtypes. Desensitization by multiple protein kinases via receptor phosphorylation is pathway selective. Structural determination of 5-HT1 receptor and G-protein domains that mediate G-protein-specific coupling and desensitization could lead to the development of highly selective ligands that directly regulate receptor-G-protein coupling.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, Serotonin/metabolism , Signal Transduction/physiology , Animals , GTP-Binding Proteins/metabolism , Humans , Receptors, Serotonin, 5-HT1 , Sensitivity and Specificity
9.
Curr Biol ; 11(6): 405-16, 2001 Mar 20.
Article in English | MEDLINE | ID: mdl-11301251

ABSTRACT

BACKGROUND: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the alpha subunits of classical G(i)-type G proteins. RESULTS: To investigate the possibility that G protein betagamma subunits, rather than alpha subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein betagamma scavengers (G(t)alpha and betaARK-C(CAAX)) to sequester free Gbetagamma dimers. We demonstrated that the injection of mRNA encoding either of these Gbetagamma scavengers induced oocyte maturation. The Gbetagamma scavengers bound an endogenous, membrane-associated Gbeta subunit, indistinguishable from Xenopus Gbeta1 derived from mRNA injection. The injection of Xenopus Gbeta1 mRNA, together with bovine Ggamma2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation. CONCLUSION: An endogenous G protein betagamma dimer, likely including Xenopus Gbeta1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gbetagamma scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gbetagamma.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , Heterotrimeric GTP-Binding Proteins/metabolism , Meiosis/physiology , Oocytes/growth & development , Oogenesis/physiology , Animals , Cyclic AMP/metabolism , Female , Gene Expression , Humans , Oocytes/drug effects , Progesterone/pharmacology , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Xenopus laevis
10.
Synapse ; 37(2): 125-45, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10881034

ABSTRACT

Dopamine D(5) receptor localization has been difficult because even the most specific ligands cannot distinguish between molecular subtypes of the D(1)-like receptor subfamily. Antifusion protein rabbit polyclonal antibodies directed against the C-terminus of human D(5) receptor were therefore developed for immunolocalization of the D(5) receptor protein in brain. The antibodies were characterized by immunoblot analysis and immunoprecipitation and used for light microscopic immunocytochemistry in rat and monkey brain. Affinity purified D(5) antibodies were specific for D(5) fusion protein as well as cloned and native D(5) receptor on Western blots, and D(5) antisera specifically immunoprecipitated solubilized, cloned D(5) receptor. Regional distribution of D(5) receptor immunoreactivity was consistent across species and correlated well with D(5) mRNA distribution previously reported in monkey brain. Immunoreactivity was widespread and tended to label perikarya and proximal dendrites of neurons in cerebral cortex, basal ganglia, basal forebrain, hippocampus, diencephalon, brainstem, and cerebellum. Neuropil was immunoreactive in olfactory bulb, islands of Calleja, cerebral cortex, superior colliculus, and molecular layer of cerebellum. The distribution of D(5) in brain was clearly different from that of other dopamine receptor subtypes, including D(1), the other member of the D(1)-like receptor subfamily. This unique distribution corroborates the idea that the D(5) receptor subtype has a distinct role in dopamine neurotransmission.


Subject(s)
Brain/metabolism , Haplorhini/metabolism , Rats/metabolism , Receptors, Dopamine D1/metabolism , Animals , Immunoblotting , Immunohistochemistry , Male , Precipitin Tests , Receptors, Dopamine D5 , Tissue Distribution
11.
FEBS Lett ; 470(2): 183-8, 2000 Mar 24.
Article in English | MEDLINE | ID: mdl-10734231

ABSTRACT

To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist-mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding.


Subject(s)
Receptors, Dopamine D1/chemistry , Receptors, Dopamine D1/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Adenylyl Cyclases/metabolism , Antipsychotic Agents/metabolism , Antipsychotic Agents/pharmacology , Binding Sites , Cell Line , Cell Membrane/metabolism , Cyclic AMP/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Antagonists/metabolism , Dopamine Antagonists/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Gene Expression , Humans , Ligands , Receptors, Dopamine D1/agonists , Receptors, Dopamine D1/genetics , Receptors, Dopamine D5 , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/genetics , Structure-Activity Relationship , Thermodynamics , Transfection
12.
J Biol Chem ; 274(45): 31882-90, 1999 Nov 05.
Article in English | MEDLINE | ID: mdl-10542214

ABSTRACT

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.


Subject(s)
Receptors, Dopamine D1/metabolism , Amino Acid Sequence , Antipsychotic Agents/metabolism , Benzazepines/metabolism , Binding, Competitive , Cell Line , Dopamine/metabolism , Humans , Kinetics , Molecular Sequence Data , Protein Conformation , Receptors, Dopamine D1/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship
13.
Tumour Biol ; 19(2): 113-7, 1998.
Article in English | MEDLINE | ID: mdl-9486562

ABSTRACT

OBJECTIVES: To evaluate the effect of terminal renal failure on the ratio of free to total prostate-specific antigen (PSA). METHODS: We measured free and total PSA in a group of 48 men on chronic hemodialysis. Samples were taken before dialysis and compared with a control group of 101 men without known prostatic or renal disease. Measurements of total and free PSA were made simultaneously with an ES-300 analyzer from Boehringer Mannheim according to the manufacturer's instructions. RESULTS: Patients on hemodialysis have lower complexed PSA but higher free PSA than controls. Therefore, we observed a markedly higher calculated proportion of free PSA in the dialysis group (0.54 +/- 0.16 vs. 0.33 +/- 0.14, p < 0.00001). The calculated proportion of free PSA decreases progressively as the levels of total PSA increases from 0 to 4 microg/l in both groups. By multivariate analysis, total PSA (p < 0.00001) and dialysis (p < 0.00001) were independent predictors of the fraction of free PSA. Age had no significant effect after correction for total PSA (p = 0.23). CONCLUSION: Patients on hemodialysis have normal total PSA but a significantly higher proportion of free PSA. Caution is therefore recommended for the interpretation of free PSA in that population since cutoff established in patients without renal disease may be inappropriate.


Subject(s)
Kidney Failure, Chronic/blood , Kidney Failure, Chronic/therapy , Prostate-Specific Antigen/blood , Renal Dialysis , Autoanalysis , Creatinine/blood , Humans , Male , Middle Aged , Probability , Reference Values , Urea/blood
14.
J Biol Chem ; 271(45): 28071-6, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8910419

ABSTRACT

Recently, we have shown that the dopamine D1B/D5 receptor displays binding and coupling properties that are reminiscent of those of the constitutively activated G protein-coupled receptors when compared with the related D1A/D1 receptor subtype (Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931). The carboxyl-terminal region of the third cytoplasmic loop of several G protein-coupled receptors has been demonstrated to be important for the regulation of the equilibrium between inactive and active receptor conformations. In this cytoplasmic region, the primary structure of dopamine D1A and D1B receptors differs by only two residues: Phe264/Arg266 are present in D1A receptor compared with Ile288/Lys290 in the D1B receptor. To investigate whether these structural differences could account for the distinct binding and coupling properties of these dopamine receptor subtypes, we swapped the variant residues located in the carboxyl-terminal region by site-directed mutagenesis. The exchange of the D1A receptor residue Phe264 by the D1B receptor counterpart isoleucine led to a D1A receptor mutant exhibiting D1B-like constitutive properties. In contrast, substitution of D1B receptor Ile288 by the D1A receptor counterpart phenylalanine resulted in a loss of constitutive activation of the D1B receptor with binding and coupling properties similar to the D1A receptor. The Arg/Lys substitution had no effect on the function of either receptor. These results demonstrate that the carboxyl-terminal region, and in particular residue Ile288, is a major determinant of the constitutive activity of the dopamine D1B receptor. Moreover, these results establish that not only can agonist-independent activity of a receptor be induced, but when given the appropriate mutation, it can be reversed or silenced.


Subject(s)
Receptors, Dopamine D1/metabolism , Adenylyl Cyclases/metabolism , Arginine , Benzazepines/analogs & derivatives , Benzazepines/metabolism , Binding, Competitive , Butaclamol/metabolism , Cyclic AMP/metabolism , Dopamine/administration & dosage , Dopamine/metabolism , Dose-Response Relationship, Drug , Humans , Isoleucine , Models, Molecular , Phenylalanine , Protein Conformation , Receptors, Dopamine D1/genetics , Receptors, Dopamine D5 , Structure-Activity Relationship
15.
J Biol Chem ; 271(7): 3771-8, 1996 Feb 16.
Article in English | MEDLINE | ID: mdl-8631993

ABSTRACT

The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Dopamine D1/physiology , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Cell Line , Cyclic AMP-Dependent Protein Kinases/biosynthesis , Dopamine/pharmacology , Enzyme Activation , G-Protein-Coupled Receptor Kinase 3 , G-Protein-Coupled Receptor Kinase 5 , Humans , Kidney , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Rats , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptors, Dopamine D1/biosynthesis , Recombinant Proteins/biosynthesis , Sequence Tagged Sites , Transfection , beta-Adrenergic Receptor Kinases , beta-Galactosidase/biosynthesis
16.
J Biol Chem ; 269(45): 27925-31, 1994 Nov 11.
Article in English | MEDLINE | ID: mdl-7525564

ABSTRACT

Dopamine D1A and D1B receptor subtypes belong to the superfamily of G protein-coupled receptors. Both receptors are coupled to the activation of adenylyl cyclase and exhibit distinct brain distribution. To identify functional differences, binding and stimulation of adenylyl cyclase were assessed in 293 cells expressing transiently either dopamine D1A or D1B receptors. Membranes expressing D1B receptors displayed higher affinities for agonists than those expressing D1A receptors, whereas antagonist affinities were lower at the D1B than at the D1A receptor. Basal activity of adenylyl cyclase in whole 293 cells expressing various levels of D1B receptors was significantly higher than the basal activity measured in cells expressing D1A receptors. Maximal activation of adenylyl cyclase resulting from stimulation of the D1B receptor was less than that obtained following agonist activation of the D1A receptor. In cells expressing D1B receptors, agonists displayed an increased potency for stimulating adenylyl cyclase in comparison with the potencies determined for the D1A receptor. On the other hand, certain antagonists displayed "negative efficacy" at both receptor subtypes but had a more profound inhibition on the agonist-independent signaling activity of the D1B receptor. The properties described here are reminiscent of those of constitutively active G protein-coupled receptors obtained by site-directed mutations. Thus, the D1B receptor may represent a naturally occurring receptor subtype with properties akin to those of constitutively active G protein-coupled receptors. The different anatomical distribution and biochemical properties of these D1 receptors strengthen the functional distinctions between the two subtypes and could account for the basis of heterogeneity within a given class of neurotransmitter or hormone receptors. In addition, if these properties are recapitulated in cells expressing the D1B receptors, they may underlie important role in the regulation of physiological effects by dopamine. Finally, these results raise the interesting possibility that psychotropic antagonist drugs used in the management of certain brain disorders may have their beneficial actions as negative efficacy compounds.


Subject(s)
Adenylyl Cyclases/metabolism , Brain/metabolism , Cyclic AMP/metabolism , Receptors, Dopamine D1/agonists , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Animals , Apomorphine/pharmacology , Benzazepines/pharmacology , Butaclamol/pharmacology , Cell Line , Cell Membrane/metabolism , Dopamine/pharmacology , Dose-Response Relationship, Drug , Embryo, Mammalian , Fenoldopam/pharmacology , Flupenthixol/pharmacology , GTP-Binding Proteins/metabolism , Genomic Library , Humans , Kidney , Kinetics , Rats , Receptors, Dopamine D1/biosynthesis , Receptors, Dopamine D1/metabolism , Transfection
17.
Proc Natl Acad Sci U S A ; 91(9): 3633-6, 1994 Apr 26.
Article in English | MEDLINE | ID: mdl-8170959

ABSTRACT

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.


Subject(s)
GTP-Binding Proteins/physiology , Protein Kinases/metabolism , Receptors, Adrenergic, beta/metabolism , Animals , Cattle , Cell Membrane/metabolism , Humans , In Vitro Techniques , Recombinant Proteins , Signal Transduction , Substrate Specificity
18.
J Biol Chem ; 269(4): 2790-5, 1994 Jan 28.
Article in English | MEDLINE | ID: mdl-7507928

ABSTRACT

An aromatic residue, tyrosine 326 in the prototypical human beta 2-adrenergic receptor, exists in a highly conserved sequence motif in virtually all members of the G protein-coupled receptor family. The potential role of this conserved aromatic amino acid residue in the cellular processes of sequestration (a rapid internalization of the surface receptor) and down-regulation (a slower loss of total cellular receptors) associated with agonist-mediated desensitization of the beta 2-adrenergic receptor was assessed by replacing tyrosine residue 326 with an alanine residue (beta 2AR-Y326A). This mutation completely abolishes agonist-mediated receptor sequestration without affecting the ability of the receptor to activate maximally adenylyl cyclase, to undergo rapid desensitization, and to down-regulate in response to agonist. The only other major change associated with the mutated receptor is a complete loss of the ability to resensitize following rapid desensitization. These results imply that this tyrosine residue, which is part of a highly conserved sequence motif in G protein-coupled receptors, may be responsible for their agonist-mediated sequestration and that sequestration and down-regulation of the receptor are dissociable phenomena. The lack of resensitization in the sequestration-defective beta 2-adrenergic receptor mutant strongly suggests that the sequestration pathway is an important mechanism by which cells re-establish the normal responsiveness of G protein-coupled receptors following the removal of agonist.


Subject(s)
Conserved Sequence , GTP-Binding Proteins/metabolism , Isoproterenol/pharmacology , Protein Conformation , Receptors, Adrenergic, beta-2/metabolism , Receptors, Cell Surface/metabolism , Tyrosine , Alanine , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Down-Regulation , Epitopes/analysis , GTP-Binding Proteins/chemistry , Humans , Iodocyanopindolol , Kinetics , Molecular Sequence Data , Multigene Family , Mutagenesis, Site-Directed , Pindolol/analogs & derivatives , Pindolol/metabolism , Polymerase Chain Reaction , Radioligand Assay , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/drug effects , Receptors, Cell Surface/chemistry , Sequence Homology, Amino Acid , Transfection
19.
J Recept Res ; 13(1-4): 573-90, 1993.
Article in English | MEDLINE | ID: mdl-8450505

ABSTRACT

The sub-family of dopamine D1-like receptors is now known to be comprised of at least two members: the originally cloned D1 receptor (herein referred to as the D1a receptor) and a related receptor referred to as the D1b, D1 beta or D5 dopamine receptor (herein referred to as the D1b/D5 receptor). Here, we characterize the D1b/D5 receptor expressed transiently in COS-7 cells and permanently in Ltk- cells. Transiently expressed human D1b/D5 receptors bind the D1 specific ligand [125I]SCH 23982 saturably and with high affinity (KD = 500 pM). Competition for [125I]SCH 23982 binding to rat D1b/D5 and human D1a and D1b/D5 receptors supports the contention that the two D1b/D5 receptors are species homologues. Furthermore, in COS-7 cells, as previously observed, dopamine competes for the binding of [125I]SCH 23982 to human D1b/D5 receptors with a higher affinity than that seen at the human D1a receptor. These results are similar to those seen in Ltk- cells permanently transfected with the human D1b/D5 receptor. In these cells, dopamine competition for [125I]SCH 23982 binding is complex, sensitive to guanine nucleotides and of a higher affinity than that observed for dopamine binding to the human D1a receptor expressed in these same cells. In both D1a and D1b/D5 expressing Ltk- cells, dopamine stimulates adenylyl cyclase with an EC50 of approximately 200 nM. Furthermore, preincubation of Ltk- cells expressing the D1a and D1b/D5 receptors with dopamine results in desensitization of the response of adenylyl cyclase to subsequent agonist stimulation.


Subject(s)
Receptors, Dopamine D1/metabolism , Receptors, Dopamine/metabolism , Adenylyl Cyclases/drug effects , Animals , Binding, Competitive , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Humans , Receptors, Dopamine/drug effects , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D5 , Recombinant Proteins/metabolism
20.
FEBS Lett ; 312(2-3): 115-22, 1992 Nov 09.
Article in English | MEDLINE | ID: mdl-1426240

ABSTRACT

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.


Subject(s)
Carrier Proteins/metabolism , Central Nervous System/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cattle , Central Nervous System/chemistry , Cloning, Molecular , DNA , Molecular Sequence Data , RNA, Messenger/genetics , Rats , Sequence Homology, Amino Acid
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