Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

ZNRD1 (zinc ribbon domain-containing 1) is a host cellular factor that influences HIV-1 replication and disease progression.

BACKGROUND: Human immunodeficiency virus (HIV) takes advantage of multiple host proteins to support its own replication. The gene ZNRD1 (zinc ribbon domain-containing 1) has been identified as encoding a potential host factor that influenced disease progression in HIV-positive individuals in a genomewide association study and also significantly affected HIV replication in a large-scale in vitro short interfering RNA (siRNA) screen. Genes and polymorphisms identified by large-scale analysis need to be followed up by means of functional assays and resequencing efforts to more precisely map causal genes. METHODS: Genotyping and ZNRD1 gene resequencing for 208 HIV-positive subjects (119 who experienced long-term nonprogression [LTNP] and 89 who experienced normal disease progression) was done by either TaqMan genotyping assays or direct sequencing. Genetic association analysis was performed with the SNPassoc package and Haploview software. siRNA and short hairpin RNA (shRNA) specifically targeting ZNRD1 were used to transiently or stably down-regulate ZNRD1 expression in both lymphoid and nonlymphoid cells. Cells were infected with X4 and R5 HIV strains, and efficiency of infection was assessed by reporter gene assay or p24 assay. RESULTS: Genetic association analysis found a strong statistically significant correlation with the LTNP phenotype (single-nucleotide polymorphism rs1048412; [Formula: see text]), independently of HLA-A10 influence. siRNA-based functional analysis showed that ZNRD1 down-regulation by siRNA or shRNA impaired HIV-1 replication at the transcription level in both lymphoid and nonlymphoid cells. CONCLUSION: Genetic association analysis unequivocally identified ZNRD1 as an independent marker of LTNP to AIDS. Moreover, in vitro experiments pointed to viral transcription as the inhibited step. Thus, our data strongly suggest that ZNRD1 is a host cellular factor that influences HIV-1 replication and disease progression in HIV-positive individuals.

C3-symmetric peptide scaffolds are functional mimetics of trimeric CD40L.

Interaction between CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ligand CD40L, a 39-kDa glycoprotein, is essential for the development of humoral and cellular immune responses. Selective blockade or activation of this pathway provides the ground for the development of new treatments against immunologically based diseases and malignancies. Like other members of the TNF superfamily, CD40L monomers self-assemble around a threefold symmetry axis to form noncovalent homotrimers that can each bind three receptor molecules. Here, we report on the structure-based design of small synthetic molecules with C3 symmetry that can mimic CD40L homotrimers. These molecules interact with CD40, compete with the binding of CD40L to CD40, and reproduce, to a certain extent, the functional properties of the much larger homotrimeric soluble CD40L. Architectures based on rigid C3-symmetric cores may thus represent a general approach to mimicking homotrimers of the TNF superfamily.

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

Characterization of functional role of α-ketoglutarate receptor in the kidney : role of the intrinsic circadian timing system in renin expressing cells

Le rein joue un rôle essential dans le maintien de l'homéostasie des fluides extracellulaires (FEC) et la pression artérielle. L'objectif de notre groupe est d'identifier de nouveaux mécanismes impliqués dans le contrôle de l'homéostasie des FEC et de la pression artérielle par le rein. Projet 1) Caractérisation du rôle fonctionnel du récepteur à l'a-cétogluatarate Oxgrl dans le rein Oxgrl est le récepteur spécifique de l'a-cétogluatarate, une moléule intermédiaire du cycle de l'acide citrique, filtrée par le rein et réabsorbée ou secrétée au niveau des tubules proximaux. Le rôle fonctionnel de ces deux récepteurs reste inconnu. Nos résultats montrent qu'Oxgrl est localisé au niveau des cellules intercalaires du tube collecteur (CCD). Des souris (Oxgrr/_) montrent une diminution du pH urinaire ,une augmentation de la concentration de l'acide urinaire titrable et une augmentation des niveaux d'a-cétoglutarate. Le traitement au Na-bicarbonate provoque une augmentation plus prononcée de l'alcalose métabolique chez les souris Oxgrl"7"' accompagnée d'une augmentation de la concentration de bicarbonate et une diminution du niveau de chlore plasmatique. En parallèle, des études de microperfusion ont montré que a-cétoglutarate stimule la réabsorption éléctroneutre de NaCl dans le CCD des souris de type sauvage mais pas des souris Oxgrl"7". En résumé, ces résultats montrent que l'a-cétoglutarate joue un rôle de molécule messagère du tubule proximal jusqu'au tube collecteur au niveau du rein et qu'Oxgrl pourrait être impliqué dans la régulation de l'échange Cl/bicarbonate et la réabsorption du NaCl dans les cellules intercalées. Projet 2) Rôle du système circadien dans les cellules productrices de rénine. Le système chronologique circadien est un mécanisme moléculaire ubiquitaire qui permet à l'organisme de coordonner ses fonctions principales en fonction du temps géophysique. Comme l'activité de la rénine plasmatique montre une rythmicité circadienne nette chez l'homme et la souris ; dans ce projet, nous avons abordé la question à savoir dans quelle mesure le système circadien est impliqué dans cette variabilité circadienne. Pour cela, le gène Bmall, élément principal de l'horloge moléculaire, a été perturbé dans les cellules granulaires productrices de rénine par le système Cre-LoxP. Nos résultats montrent que les souris Renld- Cre/Bmalllox/lox (cKO) présentent de faibles taux d'ARNm de Reni, altèrent la dynamique d'expression de la protéine rénine, mais il le niveau de concentration plasmatique de la rénine reste le même. Cependant, les souris cKO montrent une réduction significative de la concentration plasmatique de l'aldostérone. Nos analyses de l'urine récupérée dans des intervalles de temps de 24 et 1 heure montrent une augmentation du volume urinaire, une tendance à une hypercalciurie, ainsi qu'une altération de la dynamique d'excrétion urinaire de sodium chez les souris cKO. Plusieurs gènes impliqués dans la production/sécrétion de la rénine et dans le contrôle de la fonction rénale montrent une altération de l'expression circadienne d'ARNm. Par ailleurs, les souris cKO montrent une baisse significative de la pression artérielle. Nos résultats suggèrent que l'horloge intrinsèque des cellules productrices de la rénine joue un rôle important dans le control des FEC et l'homéostasie de la pression artérielle via régulation de la fonction rénale.

Identification and functional characterization of Rca1, a transcription factor involved in both antifungal susceptibility and host response in Candida albicans.

The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response.

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis.

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.

Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Label-free cytotoxicity screening assay by digital holographic microscopy

Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

Contrasting the functional properties of GABAergic axon terminals with single and multiple synapses in the thalamus

Diverse sources of GABAergic inhibition are a major feature of cortical networks, but distinct inhibitory input systems have not been systematically characterized in the thalamus. Here, we contrasted the properties of two independent GABAergic pathways in the posterior thalamic nucleus of rat, one input from the reticular thalamic nucleus (nRT), and one "extrareticular" input from the anterior pretectal nucleus (APT). The vast majority of nRT-thalamic terminals formed single synapses per postsynaptic target and innervated thin distal dendrites of relay cells. In contrast, single APT-thalamic terminals formed synaptic contacts exclusively via multiple, closely spaced synapses on thick relay cell dendrites. Quantal analysis demonstrated that the two inputs displayed comparable quantal amplitudes, release probabilities, and multiple release sites. The morphological and physiological data together indicated multiple, single-site contacts for nRT and multisite contacts for APT axons. The contrasting synaptic arrangements of the two pathways were paralleled by different short-term plasticities. The multisite APT-thalamic pathway showed larger charge transfer during 50-100 Hz stimulation compared with the nRT pathway and a greater persistent inhibition accruing during stimulation trains. Our results demonstrate that the two inhibitory systems are morpho-functionally distinct and suggest and that multisite GABAergic terminals are tailored for maintained synaptic inhibition even at high presynaptic firing rates. These data explain the efficacy of extrareticular inhibition in timing relay cell activity in sensory and motor thalamic nuclei. Finally, based on the classic nomenclature and the difference between reticular and extrareticular terminals, we define a novel, multisite GABAergic terminal type (F3) in the thalamus.

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.