Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells.

The nucleotide sequence of a particular T1 oligonucleotide found in 41S and 28S RNAs of several cellular cell lines (human, mouse, rat and chicken fibroblast) but absent in 45S ribosomal RNA has been deduced. Its primary structure : A-U-U*-G*-psi-U-C-A-C-C-C-A-C-U-A-A-U-A-Gp shows the presence of a modified G residue which explains the existence of this oligonucleotide in the T1 fingerprint of 41S RNA and 28S. Its absence on the 45S RNA T1 fingerprint is accounted for by a late modification.     

SLC26A4 Targeted to the Endolymphatic Sac Rescues Hearing and Balance in Slc26a4 Mutant Mice

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 ^Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 ^Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.     

Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.     

Sequence determination of 5'-terminal and 3'-terminal T1 oligonucleotides of 18-S ribosomal RNA of a mouse cell line (L 5178 Y).

The 5' and 3'-terminal oligonucleotides of 18-S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1-ribonuclease-hydrolysed 18-S RNA. These two oligonucleotides were partially hydrolysed by snake venom and spleen phsophodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU-A-C-C-U-G for the 5'-terminal oligonucleotide and G-A-U-C-A-U-U-Aoh for the 3'-terminal oligonucleotide. Quantitative studies indicated that these sequences represent at least 70% for the 5' oligonucleotide and 85% for the 3' oligonucleotide of the terminal sequences of the 18-S RNA.     

Sequence determination of the 3'' terminal T1 oligonucleotide of 18S ribosomal RNA.

We have reexamined the primary structure of the 3' terminal oligonucleotide of 18S RNA from chicken fibroblasts and have shown, contrary to previously published results that this extremity G-A-U-C-A-U-U-AOH is identical to that of the rabbit, drosophila and bombyx. Furthermore the electrophoretic mobility and composition of the 3' terminal oligonucleotides of 18S RNA from rat and human cells are similar to that of other RNAs and show that the identity of structure for this region of 18S RNA extends to include all tested species between yeast and man. This finding reveals a marked degree of evolutionary constraint on the structure of this region.     

Insulin receptor-related receptor as an extracellular pH sensor involved in the regulation of acid–base balance

Recent studies of insulin receptor-related receptor (IRR) revealed its unusual property to activate upon extracellular application of mildly alkaline media, pH > 7.9. The activation of IRR with hydroxyl anion has typical features of ligand–receptor interaction; it is specific, dose-dependent, involves the IRR extracellular domain and is accompanied by a major conformational change. IRR is a member of the insulin receptor minifamily and has been long viewed as an orphan receptor tyrosine kinase since no peptide or protein agonist of IRR was found. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin-like growth factor receptors in amphibia. The latter two cannot be activated by alkali. Another major difference between them is that unlike ubiquitously expressed insulin and insulin-like growth factor receptors, IRR is found in specific sets of cells of only some tissues, most of them being exposed to extracorporeal liquids of extreme pH. In particular, largest concentrations of IRR are in beta-intercalated cells of the kidneys. The primary physiological function of these cells is to excrete excessive alkali as bicarbonate into urine. When IRR is removed genetically, animals loose the property to excrete bicarbonate upon experimentally induced alkalosis. In this review, we will discuss the available in vitro and in vivo data that support the hypothesis of IRR role as a physiological alkali sensor that regulates acid–base balance. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

A mutant alphaII-spectrin designed to resist calpain and caspase cleavage questions the functional importance of this process in vivo

alpha- and beta-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous alphaII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of alphaII-spectrin by these enzymes could be involved in important functions. To assess the role of alphaII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant alphaII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant alphaII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular alphaII-spectrin cleavage in either major cellular functions or in normal development.     

Defective ENaC processing and function in tissue kallikrein-deficient mice

An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK-/-) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of gamma-ENaC in TK-/- mice. Urinary Na+ excretion after amiloride injection was blunted in TK-/- mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK-/- mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of gamma-ENaC was increased, indicating that its absence in TK-/- mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK-/- mice with TK resulted in the appearance of the 70-kDa band of the gamma-ENaC, indicating that TK was able to promote gamma-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon.