F. de Sauvage

Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

Heat‐stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl‐ secretion, and that this is abolished by inhibitors of cAMP‐dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl‐ secretion only in cells expressing the wild‐type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa‐induced diarrhea.

Alternative polyadenylation of the amyloid protein precursor mRNA regulates translation.

The sequence of several cDNAs encoding the amyloid protein precursor showed that two polyadenylation sites of the mRNA are utilized; RNA blot analysis with different riboprobes indicated that this explains the difference between the two major 3.2 and 3.4 kb mRNAs found in the human brain. These two mRNAs, which contain the whole sequence of the natural molecules, were synthesized by in vitro transcription and translated in Xenopus oocytes. The long mRNA using the second polyadenylation site produced more protein than the short mRNA. The sequence contained within the two polyadenylation sites used in the 3′ untranslated region of the amyloid protein precursor mRNA was also able to increase the production of the chicken lysozyme or the chloramphenicol acetyl transferase, as demonstrated by in vivo translation of different chimeric mRNAs obtained by in vitro transcription. This difference in protein production was also observed when chimeric cDNA constructs were transfected into Chinese hamster ovary cells. Since long mRNAs are not more stable than short mRNAs, the sequence contained within the two polyadenylation sites of the amyloid protein precursor mRNA increases the translation.

Modification of neuronal cell adhesion affects the genetic expression of the A4 amyloid peptide precursor.

Heparan sulfate proteoglycans play a key role in neuronal cell adhesion and their core protein share amino acid sequence with the A4 amyloid peptide precursor. Modification of neuronal cell adhesion by heparin induces important morphological changes in rat cultured neurones and increases the genetic expression of the A4 amyloid peptide precursor.

Cloning of the cDNA from normal brain and brain of patients with Alzheimer's disease in the expression vector lambda GT 11.

1. RNA was purified from postmortem human brains, and the poly A+ RNA was isolated by oligo dT cellulose. 2. Double stranded cDNA was synthesized using reverse transcriptase, RNAse H and DNA polymerase. 3. cDNA was cloned in the lambda GT 11 expression vector, and libraries containing between 1 and 2 millions clones were obtained. 92 to 98% of the plaques contained a recombinant phage. 4. Such libraries will allow the molecular characterization of cDNA and corresponding proteins which play a key role in brain functions and in particular which could be involved in the etiology of Alzheimers dementia.

Identification of different β amyloid cDNAs cloned from the brain of a patient with sporadic alzheimer's disease☆

Neurofibrillary tangles and senile plaques, two neuropathological markers of Alzheimers disease, may both contain peptide fragments derived from the ? amyloid protein. Human ? amyloid peptide precursor cDNAs have been isolated from normal foetal and adult brain libraries. In peripheral tissue and cultured cells, a novel precursor containing a protease inhibitor domain has been cloned. A cDNA library from the cerebral cortex of a patient with sporadic Alzheimers disease was constructed and several clones coding for the ? amyloid peptide precursor were isolated cDNAs containing two types of insertion coding for a serine protease inhibitor domain were identified. The use of another polyadenylation site available in the 3?-untranslated region of the mRNA was observed. These results indicate that, in one patient with Alzheimers disease, different RNA species coding for the ? amyloid peptide precursor arise by alternative splicing of a single transcriptional unit, and use different polyadenylation sites.

CONSTRUCTION OF LAMBDA GT 11 cDNA LIBRARIES FROM POST-MORTEM HUMAN BRAINS

ABSTRACT The lambda gt 11 expression vector has been used in order to construct cDNA libraries from human brain RNA. If brain suffering and post-mortem delay are not too long, RNA was able to produce high molecular weight proteins when translated in a rabbit reticulocyte lysate. The mRNA was transformed into cDNA in order to be cloned in the lambda vector. After in vitro packaging, libraries were obtained, containing between 106 and 2.106 plaques, 92 to 98% of them being recombinants. cDNA libraries from normal cerebral cortex, caudate nucleus, cerebellum, hippocampus and substancia nigra as well as from Alzheimers brain have been constructed.

The sequence within the two polyadenylation sites of the A4 amyloid peptide precursor stimulates the translation

cDNA probes specific for the A4 amyloid peptide precursor hybridize with a 3.2–3.4 kb mRNA doublet which can be attributed to the use of two polyadenylation sites. Different chimeric mRNAs were synthesized by in vitro transcription of the coding region of the chicken lysozyme or the chloramphenicol acetyl transferase followed by two 3′ untranslated regions of the A4 amyloid peptide precursor mRNA using the two possible polyadenylation sites. In vivo translation of these mRNA constructs in Xenopus oocytes indicates that the long mRNAs using the second polyadenylation site produce higher amounts of proteins as compared to the short mRNAs. This effect on the translation is not related to a higher stability of the long mRNA in oocytes.