Biomedical subjects

Laura Conforti

Publications and source records attributed to Laura Conforti.

23 records · Page 2Linked to original sources

PubMed2002

Expression of the Na+-HCO-3 cotransporter NBC4 in rat kidney and characterization of a novel NBC4 variant.

The purpose of the present studies was to examine the renal distribution and functional properties of Na(+)-HCO(3)(-) cotransporter type 4 (NBC4), the latest NBC isoform to be identified. Zonal distribution studies in rat kidney by Northern blot hybridization and RT-PCR demonstrated that NBC4 is highly abundant in the outer medulla and cortex but is low in the inner medulla. Nephron segment distribution studies indicated that NBC4 is predominantly expressed in the medullary and cortical thick ascending limb of the loop of Henle. Using specific primers on the basis of the published sequence (GenBank accession no. AF-207661), a full-length NBC4 variant was cloned from human liver and examined. The sequence of this variant (called NBC4e) is shorter by 86 amino acids vs. the published sequence. Xenopus laevis oocytes injected with the full-length NBC4e cRNA were compared with NBC1-expressing oocytes. Although exposure of NBC1-expressing oocytes to CO(2)/HCO(3)(-) resulted in immediate hyperpolarization, the NBC4-expressing oocytes did not show any alteration in membrane potential. NBC activity in oocytes, assayed as the Na(+)-dependent, HCO(3)(-)-mediated intracellular pH recovery from acidosis, indicated that NBC4 is a DIDS-inhibitable NBC. We propose that NBC4 is expressed in the thick ascending limb of the loop of Henle and mediates cellular HCO(3)(-) uptake in this segment.

Animals↗

PubMed2002

Palmitoylation of KChIP splicing variants is required for efficient cell surface expression of Kv4.3 channels.

The Ca(2+)-binding proteins KChIP1-4 (KChIP3 is also known as DREAM and calsenilin) act as auxiliary subunits for voltage-gated K(+) channels in the Kv4 family. Here we identify three splicing isoforms of rat KChIP2 with variable N-terminal peptides. The two longer isoforms, which contain the 32-amino acid peptide, produce larger increases in Kv4.3 protein level and current density and more effectively localize themselves and their associated channels at the plasma membrane than the shortest variant. The 32-amino acid peptide contains potential palmitoylation cysteines. Metabolic labeling demonstrates that these cysteines in the KChIP2 isoforms, as well as the corresponding sites in KChIP3, are palmitoylated. Mutating these cysteines reduces their plasma membrane localization and the enhancement of Kv4.3 current density. Thus, palmitoylation of the KChIP auxiliary subunits controls plasma membrane localization of their associated channels.

Alternative Splicing↗

PubMed2002

Human homologue of a gene mutated in the slow Wallerian degeneration (C57BL/Wld(s)) mouse.

The slow Wallerian degeneration mouse (C57BL/Wld(s)) is a mutant strain of mouse, with the unique phenotype of prolonged survival of the distal axon following axotomy. The causative mutation is an 85 kb tandem triplication on distal mouse chromosome 4. The dominant slow Wallerian degeneration phenotype is conferred by a hybrid gene within the triplication, comprising a gene of previously unknown function, D4Cole1e, and the 5' end of ubiquitination factor E4B (Ube4b). It encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ube4b and 303 amino acids derived from the D4Cole1e gene. We have identified the human homologue of D4Cole1e, and mapped it to chromosome 1p36.2. Additional fluorescence in situ hybridisation signals indicate the presence of several homologous human sequences. Northern blot analysis shows two transcripts, widely expressed at varying levels in different human tissues. The human cDNA, which encodes a protein of 279 amino acids, has 80% nucleotide identity with the mouse cDNA. The derived human and mouse protein sequences share 78% amino acid identity and 82% amino acid similarity. The human cDNA and protein sequences are identical to the human nicotinamide mononucleotide adenylyltransferase (NMNAT). We have also determined the intron/exon structure of the gene, which will facilitate the screening of these exons for mutations in human neurodegenerative disorders.

Amino Acid Sequence↗

PubMed2002

Genotyping methods to detect a unique neuroprotective factor (Wld(s)) for axons.

The slow Wallerian degeneration mouse, C57BL/Wld(s), carries a dominant mutation that delays Wallerian degeneration in the distal stump of an injured axon. The protective gene has been identified and also found to protect axons from the neurotoxin vincristine. Therefore, it is important to determine whether it has a widespread application to protect axons in neurological disease. In principle, this can be done by crossing Wld(s) to neurological mutant mice, but first a method is needed to track the inheritance of the neuroprotective Wld(s) allele. Due to the complex nature of the mutation, there is no simple method to distinguish Wld(s) homozygotes, heterozygotes and wild-type mice. Therefore, we report a genotyping method for Wld(s) based upon pulsed field gel electrophoresis (PFGE) and compare it with the alternatives of PCR and Southern blotting. The effect of the Wld(s) mutation on axon degeneration in diverse inherited pathologies, and the consequence for symptoms, can now be investigated.

Animals↗

PubMed2002

Hypoxic vasorelaxation inhibition by organ culture correlates with loss of Kv channels but not Ca(2+) channels.

We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% N(2)-5% CO(2)) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular Ca(2+) concentration ([Ca(2+)](i)) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in K(+) and Ca(2+) channel activity and gene expression that play key roles in [Ca(2+)](i) regulation in vascular smooth muscle (VSM). Reduced mRNA expression of O(2)-sensitive K(+) channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated K(+) channels (Kv1.2 and Kv1.3) or Ca(2+) channel subtypes was found. Modified K(+) channel expression is supported by functional evidence indicating a reduced response to general K(+) channel activation, by pinacidil, and to specific voltage-dependent K(+) (Kv) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in Ca(2+)-dependent pathways of hypoxia-induced vasodilation.

Animals↗