Cadmium, copper, iron, and zinc concentrations in kidneys of grey wolves, Canis lupus, from Alaska, Idaho, Montana (USA) and the Northwest Territories (Canada).

Cadmium, copper, iron, and zinc levels were measured in the kidneys of 115 grey wolves (Canis lupus) from Idaho, Montana and Alaska (United States), and from the Northwest Territories (Canada). No significant differences in the levels of iron or copper were observed between locations, but wolf kidneys from more northern locations had significantly higher cadmium levels (Alaska > Northwest Territories > Montana ≈ Idaho), and wolves from Alaska showed significantly higher zinc than other locations. Additionally, female wolves in Alaska had higher iron levels than males, and adult wolves in Montana had higher copper levels than subadults.

Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.

17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2.

Recently a number of nonclass I genes were discovered in the human MHC class I region. One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin. FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after gamma-interferon induction. FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability. Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase. Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.

Class II transactivator-independent endothelial cell MHC class II gene activation induced by lymphocyte adhesion.

NK cells induce MHC class II molecules on the surface of allogeneic endothelial cells in an adhesion-dependent, IFN-gamma-independent manner. Here, we demonstrate that NK cells induce HLA-DR on the surface of a mutant cell line that is defective in IFN-gamma-induced MHC class II expression. RNA analysis in these cells and in a cell line that is defective in class II transactivator (CIITA) demonstrates that NK cell-induced HLA-DR alpha mRNA expression is also CIITA-independent. The Janus kinase-1-deficient cell line U4A expresses HLA-DR alpha mRNA in response to NK cell activation, and HLA-DR alpha promoter constructs transfected into these cells are induced by NK cells but not IFN-gamma. These data indicate that the IFN-gamma-independent component of the target cell HLA-DR expression induced by lymphocyte adhesion uses a signaling pathway that is distinct from the IFN-gamma-dependent mechanism and also suggest that CIITA is not required.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement.

As an adhesion receptor, the beta2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3'-untranslated region of the uPAR cDNA into a serum-inducible rabbit beta-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3'-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3' AU-rich elements.

Endoglycosidic cleavage of branched polymers by poly(ADP-ribose) glycohydrolase.

Post-translational modification of nuclear proteins with poly(ADP-ribose) modules chromatin structure and may be required for DNA processing events such as replication, repair and transcription. The polymer-catabolizing enzyme, poly(ADP-ribose) glycohydrolase, is crucial for the regulation of polymer metabolism and the reversibility of the protein modification. Previous reports have shown that glycohydrolase digests poly(ADP-ribose) via an exoglycosidic mechanism progressing from the protein-distal end of the polymer. Using two independent approaches, we investigated the possibility that poly(ADP-ribose) glycohydrolase also engages in endoglycosidic cleavage of polymers. First, partial glycohydrolase digestion of protein-bound poly(ADP-ribose) led to the production of protein-free oligomers of ADP-ribose. Second, partial glycohydrolase digestion of a fixed number of protein-free poly(ADP-ribose) polymers resulted in a transient increase in the absolute number of polymers while polymer size continuously decreased. Furthermore, endoglycosidic activity produced linear polymers from branched polymers although branch points themselves were not a preferential target of cleavage. From these data, we propose a mechanism whereby poly(ADP-ribose) glycohydrolase degrades polymers in three distinct phases; (a) endoglycosidic cleavage, (b) endoglycosidic cleavage plus exoglycosidic, processive degradation, (c) exoglycosidic, distributive degradation.

A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP.

An apparent paradox in smooth muscle biology is the ability of unphosphorylated myosin to maintain a filamentous structure in the presence of ATP in vivo, whereas unphosphorylated myosin filaments are depolymerized in vitro in the presence of ATP. This suggests that additional uncharacterized factors are required for the stabilization of myosin filaments in the presence of ATP. We report here that an abundant smooth muscle protein forms sedimentable complexes with unphosphorylated smooth muscle myosin, partially reverses the depolymerizing effect of ATP on unphosphorylated myosin, and promotes the assembly of minifilaments as revealed by electron microscopy. This protein is called kinase-related protein (KRP) because it is derived from a gene within the gene for myosin light chain kinase (MLCK) and has an amino acid sequence identical to the carboxyl-terminal domain of MLCK. Consistent with the results with purified KRP, deletion of the KRP domain within MLCK results in a diminished ability of MLCK to interact with unphosphorylated myosin. KRP binds to the heavy meromyosin fragment of myosin but not to myosin rod or fragments lacking the hinge region and light chains. Altogether, these results suggest that KRP may play a critical role in stabilizing unphosphorylated myosin filaments and that the KRP domain of MLCK may be important for subcellular targeting to filaments.