Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.