Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation.

This work describes the ab initio procedure employed to build an activation model for the alpha 1b-adrenergic receptor (alpha 1b-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. A combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha 1b-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha 1b-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.

Effect of single nucleotide polymorphisms in cytochrome P450 isoenzyme and N-acetyltransferase 2 genes on the metabolism of artemisinin-based combination therapies in malaria patients from Cambodia and Tanzania.

The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C → T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C → T and 2850C → T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

D-amphetamine fails to increase extracellular dopamine levels in mice lacking alpha 1b-adrenergic receptors: relationship between functional and nonfunctional dopamine release.

It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.

Association mapping of candidate genes for drought tolerance in Mediterranean durum wheat landraces

Treball al que se li ha concedit el premi al millor póster del congrés

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Vaniprevir with pegylated interferon alpha-2a and ribavirin in treatment-naïve patients with chronic hepatitis C: a randomized phase II study.

Vaniprevir (MK-7009) is a macrocyclic hepatitis C virus (HCV) nonstructural protein 3/4A protease inhibitor. The aim of the present phase II study was to examine virologic response rates with vaniprevir in combination with pegylated interferon alpha-2a (Peg-IFN-α-2a) plus ribavirin (RBV). In this double-blind, placebo-controlled, dose-ranging study, treatment-naïve patients with HCV genotype 1 infection (n = 94) were randomized to receive open-label Peg-IFN-α-2a (180 μg/week) and RBV (1,000-1,200 mg/day) in combination with blinded placebo or vaniprevir (300 mg twice-daily [BID], 600 mg BID, 600 mg once-daily [QD], or 800 mg QD) for 28 days, then open-label Peg-IFN-α-2a and RBV for an additional 44 weeks. The primary efficacy endpoint was rapid viral response (RVR), defined as undetectable plasma HCV RNA at week 4. Across all doses, vaniprevir was associated with a rapid two-phase decline in viral load, with HCV RNA levels approximately 3 log(10) IU/mL lower in vaniprevir-treated patients, compared to placebo recipients. Rates of RVR were significantly higher in each of the vaniprevir dose groups, compared to the control regimen (68.8%-83.3% versus 5.6%; P < 0.001 for all comparisons). There were numerically higher, but not statistically significant, early and sustained virologic response rates with vaniprevir, as compared to placebo. Resistance profile was predictable, with variants at R155 and D168 detected in a small number of patients. No relationship between interleukin-28B genotype and treatment outcomes was demonstrated in this study. The incidence of adverse events was generally comparable between vaniprevir and placebo recipients; however, vomiting appeared to be more common at higher vaniprevir doses. CONCLUSION: Vaniprevir is a potent HCV protease inhibitor with a predictable resistance profile and favorable safety profile that is suitable for QD or BID administration.

Rapid evolution of cancer/testis genes on the X chromosome.

BACKGROUND: Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole. RESULTS: To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes. CONCLUSION: Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.