cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.     

Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria.

1. Swelling of mitochondria was induced in non-respiring mitochondria by 30 mM or more Na2SO4 or by respiration in the presence of K2SO4. Respiration-drive swelling resulted in loss of respiratory control. Sulphate, when present at 10 mM concentration, promoted the release of accumulated Ca2+. 2. Swelling was prevented by N-ethylmaleimide and formaldehyde, known inhibitors of the phosphate carrier. Sulphate-induced swelling was more sensitive to the inhibitors than was phosphate-induced swelling. At lower concentration of sulphate, 5 mM, an alkalinisation of the medium was observed in addition of sulphate, indicating H+-sulphate symport. There was competition between sulphate and phosphate for transport by this mechanism. It is suggested that sulphate may be transported, though at a comparatively slow rate, by the phosphate carrier. 3. Uptake of sulphate was stimulated when preceded by energy-dependent accumulation of Ba2+, especially when acetate was also present, indicating precipitation of BaSO4 in the matrix. Using this system the influx of sulphate was studied at lower concentrations, 10 mM or less. the contributions of the H+ symporter (sensitive to N-ethylmaleimide) and the dicarboxylate carrier (sensitive to butylmalonate) could then be studied. The dicarboxylate carrier had a lower Km and was mainly responsible for sulphate transport at lower concentration range. At 10 mM-sulphate the transport rates by the two systems appeared to be similar; at still higher concentrations the H+ symporter may become more important.     

Electrophoretic transport of Tl+ in mitochondria

Summary The distribution of Tl⁺ between rat liver mitochondria and the medium was studied; millimolar or smaller concentrations of Tl⁺ were labeled with²⁰⁴Tl. The Tl⁺ distribution responded to transient diffusion potentials in a way that indicated electrophoretic movements of Tl⁺. The diffusion potentials were induced by efflux of K⁺ in response to addition of valinomycin to nonrespiring mitochondria suspended in a medium with low concentrations of K⁺ or by efflux of H⁺ induced by making the medium more alkaline in the presence of a protonophorous (proton-conducting) uncoupling agent. Changes in membrane potential induced by valinomycin were followed with the aid of safranine. Tl⁺ brought about collapse of the diffusion potential. It is concluded that Tl⁺ is able to penetrate the mitochondrial membrane electrophoretically.     

Stacking of safranine in liposomes during valinomycin-induced efflux of potassium ions.

Liposomes were prepared from phosphatidylcholine and cardiolipin in a KCl medium and suspended in a choline chloride medium with safranine. When efflux of K+ was induced by valinomycin, spectral shifts characteristic of stacking were observed. Ca2+ inhibited the rate of stacking in a competitive manner with a Ki of about 200 muM, while La3+ was about 10 times more potent. When liposomes were prepared from phospholipids with a higher ratio of cardiolipin to phosphatidylcholine the inhibition was more potent. No effect on the stacking phenomena was seen when CA2+ was added after the stacking was completed. When CA2+ or an organic cation with four charges, spermine was trapped in the intraliposomal compartment, no significant change in the rate of stacking was seen. However, the extent of stacking was decreased. It is suggested that safranine is driven by a diffusion potential to a site that is inaccessible to CA2+ in the medium, presumably to the inner boundaries of the liposomal membranes.     

Stacking of safranine in liposomes during valinomycininduced efflux of potassium ions

Liposomes were prepared from phosphatidylcholine and cardiolipin in a KCl medium and suspended in a choline chloride medium with safranine. When efflux of K⁺ was induced by valinomycin, spectral shifts characteristic of stacking were observed. Ca²⁺ inhibited the rate of stacking in a competitive manner with a K_i of about 200 μM, while La³⁺ was about 10 times more potent. When liposomes were prepared from phospholipids with a higher ratio of cardiolipin to phosphatidyl-choline the inhibition was more potent. No effect on the stacking phenomena was seen when Ca²⁺ was added after the stacking was completed. When Ca²⁺ or an organic cation with four charges, spermine, was trapped in the intraliposomal compartment, no significant change in the rate of stacking was seen. However, the extent of stacking was decreased. It is suggested that safranine is driven by a diffusion potential to a site that is inaccessible to Ca²⁺ in the medium, presumably to the inner boundaries of the liposomal membranes.     

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4⁺ T cells in the liver of infected IL27R^−/− (WSX-1^−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4⁺ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4⁺ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1^−/− mice than infected WT mice, and hepatic CD4⁺ T cells from WSX-1^−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4⁺ T cells to the liver of WSX-1^−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4⁺ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4⁺ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.