Biomedical subjects

J Buchko

Publications and source records attributed to J Buchko.

5 recordsLinked to original sources

PubMed1998

Modulation of the vascular smooth muscle angiotensin subtype 2 (AT2) receptor by angiotensin II.

The angiotensin subtype 2 (AT2) receptor is scarce in most adult vascular tissues except after injury. Since angiotensin II (AngII) is released upon injury, we examined the possibility that AngII governs AT2 receptor expression in smooth muscle cells (SMC). A polyclonal antiserum, raised to a peptide corresponding to the AT2 receptor C-terminus, recognized a approximately 45-kDa protein after transfection of cos-7 cells with AT2 receptor cDNA. Detection of a approximately 65-kDa band in extracts of SMC indicated that the AT2 receptor was glycosylated. Treatment of SMCs with AngII increased AT2 receptor levels fourfold over 24 h. This response was abrogated by losartan, but not by PD123319, indicating AT1 receptor involvement. AngII-dependent increases in AT2 receptor levels were also prevented by LY294002, an inhibitor of phosphatidyinositol 3-kinase, but not by rapamycin. These results indicate AngII influences AT2 receptor expression through the AT1 receptor via a signaling pathway that includes PI3K.

Angiotensin II↗

PubMed1998

Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.

Amino Acid Sequence↗

PubMed1998

Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current-voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being approximately 80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced ( approximately 20-40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Alternative Splicing↗

PubMed1992

The 5S ribosomal RNA gene in Pythium species: two different genomic locations.

The 5S ribosomal RNA (rRNA) genes in eukaryotes may occur either interspersed with the other rRNA genes in the ribosomal DNA (rDNA) repeat, or in separate tandem arrays, or dispersed throughout the genome. In Pythium species and in several related Oomycetes, polymerase chain reaction (PCR) amplification of the nontranscribed spacer (NTS) region with one primer specific for the 5S gene revealed, with several exceptions, that the 5S rRNA gene was present in the rDNA repeat of those species with filamentous sporangia and was absent from the rDNA repeat of those with globose or unknown sporangia. When present, the gene was located approximately 1 kb downstream of the large-subunit rRNA gene and on the strand opposite that on which the other rRNA genes were located. This was confirmed in P. torulosum by sequencing of the gene and its flanking regions. The 5S rRNA genes of P. ultimum were found in tandem arrays outside the rDNA repeat. Evidence of dispersed 5S rRNA sequences was also observed. For many of the species of Pythium having the 5S gene in the NTS, the region between the large-subunit rRNA gene and the 5S gene was found to have length heterogeneity. Oomycetes related to Pythium were also found to have the 5S gene in the NTS, although sometimes in the opposite orientation. This may mean that the presence of the gene in the NTS is ancestral for the Oomycetes and that the absence of the gene in the NTS in those Pythiums with globose sporangia is due to loss of the gene from the rDNA repeat in an ancestor of this group of species. These results favor the view that 5S rRNA gene linkage to the rRNA cistron existed prior to the unlinked arrangement seen in most plants and animals.

Base Sequence↗

PubMed1990

Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region.

About one-half of the ribosomal repeat unit of two isolates of Pythium ultimum was amplified by means of the polymerase chain reaction using one primer pair. The amplified region includes a small part of the large subunit ribosomal RNA gene, about half of the small subunit ribosomal RNA gene, and the entire intergenic region. The intergenic region of both isolates of P. ultimum has length heterogeneity due to the presence of subrepeat arrays (Klassen and Buchko 1990). PCR amplification of the heterogeneous target DNA resulted in sets of fragments which accurately reflect the heterogeneity in the target DNA, although there is a preferential amplification of the smaller targets. PCR product sizes ranged from 4.6 to 5.8 kb.

Base Sequence↗