Biomedical subjects

Tuan M Tran

Publications and source records attributed to Tuan M Tran.

11 recordsLinked to original sources

PubMed2026

Asynchronous transitions from high-risk hepatoblastoma to carcinoma.

BACKGROUND & AIMS: Most pediatric hepatocellular tumors are classified as hepatoblastoma (HB) or hepatocellular carcinoma (HCC), yet a subset exhibits mixed histological and molecular features. These hepatoblastomas with carcinoma features (HBCs) include cases provisionally designated as hepatocellular neoplasm-not otherwise specified (HCN-NOS). Their biology remains poorly understood, with unresolved questions about their cellular composition and outcomes. It is unclear whether HBCs comprise hybrid cells with combined HB and HCC characteristics (HBC cells) or admixtures of distinct HB and HCC cells. We characterized the biology, etiology, cellular composition, and evolutionary dynamics of HBCs. METHODS: We performed multi-omics profiling - including single-nucleus RNA sequencing, single-nucleus DNA sequencing, and multi-region longitudinal bulk RNA and DNA sequencing - to characterize HBC composition, evolution, and treatment response. Two-thirds of our samples were post-chemotherapy resections. RESULTS: HBCs comprise heterogeneous mixtures of HB-like, HBC-like, and HCC-like molecular cell types. Outcomes in HBC are significantly worse than in HB, and HBC cells are more chemoresistant than HB cells, with resistance shaped by their cell identity, genetic alterations, and embryonic differentiation stage. HBC cells originate from HB cells that were arrested at early hepatic stem cell development stages because of aberrant WNT signaling activation. Inhibition of WNT signaling promoted differentiation and enhanced sensitivity to chemotherapy. Furthermore, each analyzed HBC reflected a dynamic process of multiple HB-to-HBC and HBC-to-HCC transitions, underscoring their evolutionary complexity. A limitation of our study is our inability to pinpoint the role of chemotherapy-induced genome modifications. CONCLUSIONS: Multi-omics profiling of HBCs revealed key insights into their biology and composition, demonstrating that they originate from HB precursors at early hepatic stem cell development stages and that their differentiation arrest depends on sustained aberrant WNT signaling activity. IMPACT AND IMPLICATIONS: Hepatoblastomas with carcinoma features (HBCs) represent a poorly understood subset of pediatric liver tumors with mixed characteristics of hepatoblastoma (HB) and hepatocellular carcinoma (HCC). Using multi-omics profiling, we show that HBCs comprise heterogeneous mixtures of HB-like, intermediate HBC-like, and HCC-like cell populations that arise from HB precursors arrested at early hepatic stem cell developmental stages due to aberrant WNT signaling. This differentiation arrest contributes to chemoresistance and poorer clinical outcomes compared with HB. Importantly, pharmacologic inhibition of WNT signaling promoted differentiation and increased chemotherapy sensitivity, suggesting a potential therapeutic strategy. These findings refine the biological classification of HBCs and highlight differentiation-based treatment approaches for this aggressive tumor subtype.

Multiomics↗

PubMed2006

Characterization of beta2-adrenergic receptor dephosphorylation: Comparison with the rate of resensitization.

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the beta2-adrenergic receptor (beta2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t(1/2) of 9 min (k = 0.08 +/- 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 microM isoproterenol for 5 min exhibited a lag phase of approximately 5 min, after which dephosphorylation proceeded slowly with a t(1/2) of 18 min (k = 0.039 +/- 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated beta2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated beta2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 +/- 0.009/min; t(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Adenylyl Cyclases↗

PubMed2006

Salmeterol stimulation dissociates beta2-adrenergic receptor phosphorylation and internalization.

Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other beta-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting beta-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of beta-agonists. This effect may not be the same with all beta-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce beta(2)AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and beta-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant beta(2)AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-beta-arrestin2 chimera (EGFP-beta-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-beta-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit beta-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.

Adrenergic beta-2 Receptor Agonists↗

PubMed2006

Psychosis with paranoid delusions after a therapeutic dose of mefloquine: a case report.

BACKGROUND: Convenient once-a-week dosing has made mefloquine a popular choice as malaria prophylaxis for travel to countries with chloroquine-resistant malaria. However, the increased use of mefloquine over the past decade has resulted in reports of rare, but severe, neuropsychiatric adverse reactions, such as anxiety, depression, hallucinations and psychosis. A direct causality between mefloquine and severe reactions among travelers has been partly confounded by factors associated with foreign travel and, in the case of therapeutic doses of mefloquine, the central nervous system manifestations of Plasmodium infection itself. The present case provides a unique natural history of mefloquine-induced neuropsychiatric toxicity and revisits its dose-dependent nature. CASE PRESENTATION: This report describes an acute exacerbation of neuropsychiatric symptoms after an unwarranted therapeutic dose (1250 mg) of mefloquine in a 37-year-old male previously on a once-a-week prophylactic regimen. Neuropsychiatric symptoms began as dizziness and insomnia of several days duration, which was followed by one week of escalating anxiety and subtle alterations in behaviour. The patient's anxiety culminated into a panic episode with profound sympathetic activation. One week later, he was hospitalized after developing frank psychosis with psychomotor agitation and paranoid delusions. His psychosis remitted with low-dose quetiapine. CONCLUSION: This report suggests that an overt mefloquine-induced psychosis can be preceded by a prodromal phase of moderate symptoms such as dizziness, insomnia, and generalized anxiety. It is important that physicians advise patients taking mefloquine prophylaxis and their relatives to recognize such symptoms, especially when they are accompanied by abrupt, but subtle, changes in behaviour. Patients with a history of psychiatric illness, however minor, may be at increased risk for a mefloquine-induced neuropsychiatric toxicity. Physicians must explicitly caution patients not to self-medicate with a therapeutic course of mefloquine when a malaria diagnosis has not been confirmed.

Adult↗

PubMed2006

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation.

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.

Adenylyl Cyclases↗

PubMed2006

Differential phosphorylation and dephosphorylation of beta2-adrenoceptor sites Ser262 and Ser355,356.

Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.

Adrenergic beta-Agonists↗

PubMed2005

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.

Animals↗

PubMed2005

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes.

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.

Amino Acid Sequence↗

PubMed2005

Comparison of IgG reactivities to Plasmodium vivax merozoite invasion antigens in a Brazilian Amazon population.

Naturally acquired antibody reactivity to two major Plasmodium vivax vaccine candidates was investigated in 294 donors from three malaria-endemic communities of Rondônia state, Brazil. Antibody recognition of recombinantly expressed antigens covering five different regions of P. vivax reticulocyte binding protein 1 (PvRBP1) and region II of P. vivax Duffy binding protein (PvDBP-RII) were compared. Positive IgG responses to these antigens were significantly related to the level of malaria exposure in terms of past infections and years of residence in the endemic area when corrected for age. The highest prevalence of anti-PvRBP1 total IgG antibodies corresponded to the amino acid regions denoted PvRBP1(431-748) (41%) and PvRBP1(733-1407) (47%). Approximately one-fifth of positively responding sera had titers of at least 1:1,600. Total IgG responses to PvDBP-RII were more prevalent (67%), of greater magnitude, and acquired more rapidly than those to individual PvRBP1 antigens. Responses to both PvRBP1 and PvDBP-RII were biased toward the cytophilic subclasses IgG1 and IgG3. These data provide the first insights on acquired antibody responses to PvRBP1 and a comparative view with PvDBP-RII that may prove valuable for understanding protective immune responses to these two vaccine candidates as they are evaluated as components of multitarget blood-stage vaccines.

Adolescent↗

PubMed2004

Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies.

Agonist-stimulated desensitization of the beta2-adrenergic receptor (beta2AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to beta-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human beta2AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant beta2ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E1, and forskolin at maximum concentrations stimulated phosphorylation of the beta2AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still approximately 200-fold greater than for the PKA site. The EC50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the beta2AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agonist-stimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine > or = formoterol = fenoterol > terbutaline = zinterol = albuterol > salmeterol > dobutamine > or = ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous beta2AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.

Animals↗

PubMed2002

JAM2 interacts with alpha4beta1. Facilitation by JAM3.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn(2+)-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and alpha(4)beta(1) in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an alpha(4)-specific inhibitor, to attenuate the Mn(2+)-enhanced component. Intriguingly, the engagement of alpha(4)beta(1) by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/alpha(4)beta(1) interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and alpha(4)beta(1) counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on alpha(4)beta(1) interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

Cell Adhesion↗