Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary.

A 5.2-kb mRNA band that contains estrogen receptor (ER) sequence and exhibits sex- and tissue-specific expression has been identified in rat pituitary via Northern analysis; this band is composed of at least two distinctive ER mRNA isoforms. This mRNA is expressed in high levels in female pituitary but is absent in male pituitary and uterus, whereas the mRNA encoding the full-length receptor (6.2 kb) is expressed in all the aforementioned tissues. Estradiol treatment potently induces the expression of the 5.2-kb band in the male pituitary. Oligonucleotide hybridization and ribonuclease-protection experiments indicate that the pituitary ER variant is missing exons 1-4. Two corresponding cDNA clones, truncated estrogen receptor product 1 and 2 (TERP-1 and TERP-2), were isolated by using the anchored PCR. Both sequences contain a 31-bp segment of specific sequence upstream of exon 5; TERP-2, however, contains an additional 66 bp of specific sequence between the 31-bp segment and exon 5. On Northern analysis, probes complementary to the 31-bp segment of specific sequence hybridize only to the 5.2-kb band. Immunoblotting identified several proteins in rat pituitary that could represent the translation products of these or related transcripts. In summary, several ER isoforms have been identified that exhibit both tissue-specific expression and marked estrogen regulation and differ from full-length receptor by virtue of sequence upstream of the exon 4/5 boundary. Physiologically, the putative proteins encoded by these or similar isoforms might be important modulators of the tissue- and promoter-specific effects of estradiol.

British mezzotinto portraits : being a descriptive catalogue of these engravings from the introduction of the art to the early part of the present century : arranged according to the engravers, the inscriptions given at full length, and the variations of state precisely set forth : accompanied by biographical notes, and appendix of a selection of the prices produced at public sales by some of the specimens, down to the present time /

Includes indexes.

A full report of the speeches of Sir Francis Burdett at the late election : the legal arguments upon the last day of the election, particularly the admired speech of Mr. Plumer are given at full length ; together with a selection of the papers published during the election ; and a preface.

Mode of access: Internet.

A suggested production list of non-royalty plays; pt. 2. Full-length plays.

Mode of access: Internet.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris

We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. © 2006 Elsevier Inc. All rights reserved.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

Brain-derived neurotrophic factor and adenosine signalling on amyloid-β peptide induced toxicity : impact on hippocampal function

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Mécanismes moléculaires d’activation du récepteur A des peptides natriurétiques

Le récepteur A des peptides natriurétiques (NPRA) fait partie de la famille des guanylates cyclases membranaires. L’activation du NPRA par ses agonistes naturels, ANP et BNP, induit une production de GMPc qui est responsable de leur rôle dans l’homéostasie cardiovasculaire, l’inhibition de l’hypertrophie et de la fibrose cardiaques et la régulation de la lipolyse. Le NPRA est un homodimère non covalent composé d’un domaine extracellulaire de liaison du ligand (ECD), d’un unique domaine transmembranaire (TM), d’un domaine d’homologie aux kinases et d’un domaine guanylate cyclase. Bien que le NPRA ait un rôle physiologique important, les mécanismes moléculaires régissant son processus d’activation restent inconnus. Nous avons donc analysé les premières étapes du processus d’activation du NPRA. Nous avons d'abord étudié le rôle de la dimérisation des ECD dans l’activation du récepteur. Nous avons utilisé les techniques de liaison de radioligand, de FRET et de modélisation moléculaire, pour caractériser la liaison à l’ECD des agonistes naturels, d’un superagoniste et d’un antagoniste. L’ANP se lie à un dimère d’ECD préformé et la dimérisation spontanée est l’étape limitante du processus de liaison. De plus, comme le démontrent nos études de FRET, tous les peptides, incluant l’antagoniste, stabilisent le récepteur sous sa forme dimérique. Cependant, l’antagoniste A71915 stabilise le dimère d’ECD dans une conformation différente de celle induite par l’ANP. La dimérisation du NPRA semble donc nécessaire, mais non suffisante à l’activation du récepteur. L’état d’activation du NPRA dépend plutôt de l’orientation des sous unités dans le dimère. Nous avons ensuite étudié le mécanisme moléculaire de transduction du signal à travers la membrane. Plusieurs études ont suggéré que l’activation du NPRA implique un changement de conformation du domaine juxtamembranaire (JM). Cependant, les études de cristallographie de l’ECD soluble de NPRA n’ont pas permis de documenter la structure du JM et le changement de conformation impliqué dans la transduction du signal reste inconnu. Pour analyser ce changement de conformation, nous avons d’abord séquentiellement substitué les neuf acides aminés du JM par une cystéine. En étudiant la capacité des mutants à former des dimères covalents de façon constitutive ou induite par l’ANP, nous avons pu évaluer la proximité relative des résidus du JM, avant et après activation du NPRA. Ces résultats ont démontré la proximité élevée de certains résidus spécifiques et sont en contradiction avec les données cristallographiques. Nous avons également démontré que le domaine intracellulaire impose une contrainte conformationnelle au JM à l’état de base, qui est levée après liaison de l’ANP. En introduisant de 1 à 5 alanines dans l’hélice-α transmembranaire, nous avons montré qu’une rotation des TM de 40° induit une activation constitutive du NPRA. Le signal d’activation pourrait donc être transmis à travers la membrane par un mécanisme de rotation des TM. En utilisant nos données expérimentales, nous avons généré le premier modèle moléculaire illustrant la conformation active du NPRA, où les domaines JM et TM sont représentés. Dans son ensemble, cette étude apporte une meilleure compréhension des mécanismes moléculaires régissant les premières étapes du processus complexe d’activation du NPRA.

Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leptin Effects on the Regenerative Capacity of Human Periodontal Cells

Obesity is increasing throughout the globe and characterized by excess adipose tissue, which represents a complex endocrine organ. Adipose tissue secrets bioactivemolecules called adipokines, which act at endocrine, paracrine, and autocrine levels. Obesity has recently been shown to be associated with periodontitis, a disease characterized by the irreversible destruction of the tooth-supporting tissues, that is, periodontium, and also with compromised periodontal healing. Although the underlying mechanisms for these associations are not clear yet, increased levels of proinflammatory adipokines, such as leptin, as found in obese individuals, might be a critical pathomechanistic link. The objective of this study was to examine the impact of leptin on the regenerative capacity of human periodontal ligament (PDL) cells and also to study the local leptin production by these cells. Leptin caused a significant downregulation of growth (TGF beta 1, and VEGFA) and transcription (RUNX2) factors as well as matrix molecules (collagen, and periostin) and inhibited SMAD signaling under regenerative conditions. Moreover, the local expression of leptin and its full-length receptor was significantly downregulated by inflammatory, microbial, and biomechanical signals. This study demonstrates that the hormone leptin negatively interferes with the regenerative capacity of PDL cells, suggesting leptin as a pathomechanistic link between obesity and compromised periodontal healing.