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E Casanova

Publications and source records attributed to E Casanova.

15 recordsLinked to original sources
PubMed2002

Codon-improved Cre recombinase (iCre) expression in the mouse.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Animals↗
PubMed2001

A CamKIIalpha iCre BAC allows brain-specific gene inactivation.

We describe the generation of transgenic mouse lines expressing the Cre recombinase enzyme in brain under control of the CamKIIalpha gene present in a BAC expression vector. The CamKIIalpha BAC transgene gave a faithful expression pattern resembling the pattern of the endogenous CamKIIalpha gene. Specifically, high levels of CamKIIalpha Cre were detected in hippocampus, cortex, and amygdala, and lower levels were detected in striatum, thalamus, and hypothalamus. As expected, no expression was detected in the cerebellum or outside of the brain. The expression level of the BAC CamKIIalpha driven Cre was shown to be copy number dependent. To test the activity of the Cre recombinase, the transgenic mice were crossed with mice harbouring the CREB (cAMP response element binding protein) allele with the 10th exon flanked by two loxP sites, and recombination was monitored by the disappearance of the CREB protein. Finally, evaluation of the developmental postnatal expression of the CamKIIalpha Cre BAC revealed the expression of the Cre recombinase as early as P3.

Animals↗
PubMed2000

Analysis of splicing of four mouse JNK/SAPKalpha variants.

The JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) cascade is activated by a variety of stress stimuli and by the inflammatory cytokines interleukin-I (IL-I) and tumor necrosis factor alpha (TNFalpha). Four splice variants of the mouse JNK/SAPKalpha isoform, which differ in a region located in subdomains IX-X of the protein, were previously identified. Analysis of the sequence of the central region of the mouse JNK/SAPKalpha gene indicates that splice variants I and II are generated by a typical alternative splicing mechanism, while splice variants III and IV are generated by a less common mechanism, where alternative 3' splice sites located inside an exon (cryptic sites) are selected. The major splice variants alphaI and all have a wide and similar distribution in hippocampus, cerebral cortex, caudate-putamen, amygdala and the granule cell layer of cerebellum, although their expression is specifically regulated in certain cell types.

Alternative Splicing↗
PubMed1999

Inducible site-specific recombination in the brain.

The Cre/loxP recombination system allows the generation of tissue-specific somatic mutations in mice. Additional temporal control of somatic mutagenesis is highly desirable, as this would permit a more precise analysis of gene function in complex systems such as the central nervous system. Extending our previous studies, we compared several ligand-regulated recombinases, in which the ligand-binding domain (LBD) of the progesterone receptor or the estrogen receptor was fused to the Cre recombinase. A fusion protein between the Cre recombinase and a truncated LBD of the progesterone receptor was chosen to obtain inducible recombination in the brain. This fusion protein can be activated by the synthetic steroid RU486, but not by the physiological hormone progesterone. Its expression was targeted to the brain using regulatory sequences of the calcium-calmodulin-dependent kinase IIalpha or the Thy-1 gene. Application of RU486 to the mice induced Cre-mediated recombination of a lacZ reporter transgene in the cortex and hippocampus, showing that spatially and temporally controlled gene targeting can be mediated in the brain.

Animals↗
PubMed1998

Cloning of chicken and mouse alpha 1b adrenergic receptor.

A partial cDNA encoding most of the third intracellular loop of the chicken alpha 1b adrenergic receptor subtype, obtained by reverse transcription-polymerase chain reaction (RT-PCR) techniques using degenerate primers derived from mammalian sequences, was used to isolate an alpha 1b adrenergic receptor cDNA from brain. The cDNA encodes a potential protein of 507 amino acids and Northern hybridization of poly(A)+ RNA from chicken brain of different developmental stages detected a single 3.5 kb transcript. Analysis of receptor expression indicated that the alpha 1b adrenergic receptor is widely distributed in chicken tissues, specially kidney and liver. cDNA and genomic clones encoding sequences of the mouse alpha 1b adrenergic receptor were also isolated.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of the third intracellular loop of the mouse alpha1b-adrenergic receptor by cAMP-dependent protein kinase.

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. alpha1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse alpha1a-, alpha1b-, and alpha1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the alpha1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.

Amino Acid Sequence↗
PubMed1996

Identification of a long huntingtin mRNA transcript in mouse brain.

Two mRNA species of the Huntington disease (HD) gene that share identical protein coding sequences but differ in their 3' untranslated region have been identified in human. Although a similar situation has been suggested to occur in mouse, only one cDNA has been isolated to date. We report the isolation of a novel partial cDNA of the mouse HD gene that is identical in its protein coding sequence to the previously reported cDNA, although it differs in the distal portion of the 3' untranslated region. Northern blotting assays indicate that this mRNA transcript is preferentially expressed in brain, with highest levels in cerebellum, cerebral cortex and striatum.

Amino Acid Sequence↗
PubMed1996

Identification of four splice variants of the mouse stress-activated protein kinase JNK/SAPK alpha-isoform.

Four splice variants (alpha I-IV) of the stress-activated protein kinase JNK/SAPK alpha-isoform have been identified in the mouse. One of them (alpha I) contains an open reading frame of 1269 bp encoding a potential protein of 423 amino acids, whereas the second variant (alpha II) differs in a region encoding 31 amino acids located in subdomain IX. alpha III lacks this region and also differs in the terminal portion of the 3' untranslated region (3' UTR). A fourth variant (alpha IV) which lacks a region of 41 amino acids located in subdomain IX has also been identified. These splice variants are differentially expressed in mouse tissues: alpha I is the most abundant in brain areas, whereas alpha II is mainly expressed in extracerebral tissues, such as liver; alpha III and alpha IV are present in brain and other tissues although in lower amounts.

Amino Acid Sequence↗
PubMed1995

Immunodetection of serotonin transporter from mouse brain.

A DNA fragment encoding amino acid sequences from the amino terminal region of the mouse serotonin transporter was isolated and sequenced. This transporter is widely distributed throughout the mouse brain, as deduced by heminested reverse transcription-polymerase chain reaction (RT-PCR) assays. To identify the serotonin transporter protein, we have developed specific antibodies against a fusion protein containing its amino terminal region, a domain which shows a low degree of homology between the different neurotransmitter transporters. Western blot analysis of mouse brain membranes detected the serotonin transporter as a 71 kDa polypeptide.

Amino Acid Sequence↗
PubMed1995

Identification of a cyclic adenosine 3',5'-monophosphate-dependent protein kinase phosphorylation site in the carboxy terminal tail of human D1 dopamine receptor.

Each of the dopamine receptor subtypes contains several consensus sites for phosphorylation in their intracellular domains. We have used fusion proteins of the carboxy terminal tail of D1 and D5 dopamine receptors to study the phosphorylation of these proteins by cyclic adenosine 3',5' monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC). The fusion protein of D1 dopamine receptor was efficiently phosphorylated by PKA, but not by PKC. Site-directed mutagenesis of serine 380 to an alanine residue precluded the phosphorylation by the kinase. No phosphorylation of the D5 dopamine receptor fusion protein was observed with either PKA or PKC, which indicates that these receptor subtypes might differ in their mechanisms of regulation.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of alpha 1d-adrenergic receptor and tissue distribution of three alpha 1-adrenergic receptor subtypes in mouse.

A partial cDNA encoding most of the third intracellular loop of the mouse alpha 1d-adrenergic receptor subtype was amplified from hippocampus by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an alpha 1d-adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0-kb transcript. Partial cDNA fragments of the alpha 1b- and alpha 1a-adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT-PCR indicated that the alpha 1-adrenergic receptors are widely distributed in mouse tissues. The alpha 1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The alpha 1a subtype is also expressed in many tissues, whereas the alpha 1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.

Amino Acid Sequence↗
PubMed1993

Differential effect of chronic ethanol treatment on barbiturate and steroid modulation of muscimol-binding to rat brain cortex.

We report the differential alterations produced by chronic ethanol treatment on the modulation, by the barbiturate thiopental and the steroid 5 beta-pregnan-3 alpha-ol-20-one, of the binding of [3H]muscimol to membrane preparations from rat brain cortex. We found a clear barbiturate- and steroid-promoted enhancement of muscimol-binding to membranes in both control and ethanol-treated animals. However, the enhancements were higher in control animals, using the barbiturate, and in ethanol-treated rats, using the steroid, Bmax and Kd values were also differentially affected in control and ethanol-treated animals by the presence of the barbiturate or the steroid.

Animals↗
PubMed1990

Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts.

Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.

Animals↗
PubMed1979

[Immunologic studies in polymyositis syndromes].

Sixteen cases of polymyositis were submitted to study. Out of these sixteen, nine showed no clinical or paraclinical evidences of cancer, and seven presented an associated cancer. In the first group, a normal immune response was observed, whereas in the second group this response was frankly depressed. Emphasis is laid on the significance of the immune response, not only in the diagnosis but as a fundamental factor in prognosis and treatment as well. An early treatment of polymyositis without cancer proved to be satisfactory. On the other hand, when a neoplasm was present, the course of the disease was satisfactorily modified only after the extirpation of the neoplasm.

Adult↗