Chloride dependence of the K+-stimulated release of taurine from synaptosomes

Exposure of a crude synaptosomal fraction to K⁺ concentrations ranging from 25 to 100 mM evokes the release of [³H]taurine and [³H]GABA. These high concentrations of K⁺ induce, besides depolarization, a marked synaptosomal swelling, which is prevented by replacing chloride in the solutions with the largely impermeant anion gluconate. The depolarizing effect of K⁺ is unaffected by omission of chloride. The K⁺-evoked release of taurine seems related to K⁺-induced changes in synaptosomal volume rather than to a depolarizing effect, since it is totally calcium-independent but is abolished by reducing chloride and by making solutions hypertonic with mannitol. The release of [³H]GABA, in contrast is unaffected in chloride-free or hypertonic solutions.     

New method for the determination of estrogen and progesterone receptors in human breast cell lines

A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, [3H]-17 beta-Estradiol or [3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind.     

Effects of fractionated x-irradiation on the Ly-6--Ril-1--Pol-5 region

Our laboratory has focused on defining, localizing, and understanding the mode of action of genes involved in fractionated x-irradiation (FXI) leukemia in susceptible and resistant mouse strains. We have described the genetic and molecular evidence suggesting the existence of multiple independent loci involved in FXI-induced leukemogenesis. These studies indicated that one of these, Ril-1, a locus on the distal portion of chromosome 15, is the major locus influencing susceptibility to the disease. Our data unequivocally place Ril-1 in the gene complex Ly-6--Ril-1--Sis--H-30--Pol-5. Ril-1 appears to be closest to Ly-6 and Sis. We report that in FXI-induced leukemias there are hypomethylation changes in the Ly-6 region as compared to normal thymocytes. In contrast, Sis was found to be hypermethylated and not expressed. In addition, we have noted DNA rearrangements in the Ly-6--Pol-5 region in the majority of tumors examined using the Ly-6 and spleen focus-forming virus (SFFLV) molecular probes. Increased expression of Ly-6 and other surface markers encoded in this region has been noted in FXI-induced thymomas. Address correspondence and offprint request to: N. M. B. Amari.     

Unexpected inactivation of acceptor consensus splice sequence by a −3 C to T transition in intron 2 of the CFTR gene

Human Genetics - Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Analysis of DNA from a pancreatic sufficient patient by means of...     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy

Summary In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.This work was taken in part from the Ph.D. Theses of E.A. Ottinger (1994) and Q. Xu (1996), University of Minnesota, Minneapolis, MN, U.S.A. Preliminary presentations of portions of this work were made at the Twenty-Second European Peptide Symposium, Interlaken, Switzerland, September 13–19, 1992, see Ref. 1, at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995, and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995, see Ref. 2. The title side reaction was first discussed for tyrosine (see Refs 1 and 3), but all of the mechanism studies discussed herein are for serine and threonine.Amino acid symbols denote the l-configuration, and abbreviations for amino acids and peptides follow rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977].     

Taurine release associated to volume regulation in rabbit lymphocytes.

Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.     

Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.