Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3′-phosphoinositides on membrane trafficking in yeast and mammalian cells.     

Glycogen synthase kinase-3beta is involved in the process of myocardial hypertrophy stimulated by insulin-like growth factor-1.

BACKGROUND: Glycogen synthase kinase-3 beta (GSK-3beta) is involved in many cellular processes, such as metabolism, apoptosis, differentiation and proliferation. Insulin-like growth factor-1 (IGF-1), which is well known to have a hypertrophic effect on cardiomyocytes, inactivates (phosphorylates) GSK-3beta in some cell types. The role of GSK-3beta in cardiomyocytes as a negative regulator of cardiac hypertrophy has been recently reported and the present study investigated the role of GSK-3beta in the cardiac hypertrophy of cultivated neonatal rat cardiomyocytes induced by IGF-1. METHODS AND RESULTS: First, the IGF-1 induced signal transduction leading to GSK-3beta in neonatal rat cardiomyocytes was examined. The phosphatidylinositol (PI) 3-kinase/Akt/GSK-3 beta signaling induced by IGF-1 was investigated using inhibitors of PI 3-kinase and Ad AktAA, a dominant negative form of Akt. Furthermore, using Ad MEK DN, a dominant negative form of MEK, it was found that MEK negatively regulates Akt phosphorylation upon IGF-1 stimulation. Next, it was examined whether GSK-3beta acts as a negative regulator in the cardiac hypertrophy induced by IGF-1. Sustained stimulation by IGF-1 caused cardiac hypertrophy in protein synthesis and cellular morphology, and overexpression of unphosphorylatable GSK-3beta (Ad GSK-3beta S9A) repressed these hypertrophic effects of IGF-1. CONCLUSIONS: GSK-3beta may play an important role as a negative regulator of cardiac hypertrophy induced by IGF-1.     

Possible role of cytomegalovirus in the pathogenesis of inflammatory aortic diseases: a preliminary report.

To search for possible evidence of a relationship between human cytomegalovirus and aortic diseases, we examined 41 aortic lesions excised at surgery and 16 aortic tissues obtained at autopsy for the presence of cytomegalovirus DNA, by use of polymerase chain reaction. Cytomegalovirus DNA was present in seven (88%) of eight lesions of inflammatory aortic diseases with periaortic fibrosis, five of six inflammatory aneurysms, and all of two aortic occlusive lesions with inflammation. Cytomegalovirus DNA was detected in 20 (61%) of 33 atherosclerotic aneurysms, whereas it was detected in only five (31%) of 16 autopsy samples that showed neither inflammation nor atherosclerosis. Thus the possibility that cytomegalovirus may play a role in the pathogenesis of inflammatory aortic diseases warrants further attention.     

Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease

Experimental autoimmune gastritis (EAG) is an excellent model of human autoimmune gastritis, the underlying cause of pernicious anaemia. Murine autoimmune gastritis replicates human gastritis in being characterized by a chronic inflammatory mononuclear cell infiltrate in the gastric mucosa, destruction of parietal and zymogenic cells, and autoantibodies to the alpha-and beta-subunits of the gastric H+/K+ ATPase. Disease is induced strain specifically in gastritis-susceptible BALB/c mice by methods with a greater variety than those for most other experimental autoimmune diseases. The disease is induced in the regional gastric lymph node in which pathogenic CD4+ T cells are recruited. The model provides an excellent illustration of regulation by CD4+CD25+T cells, and, indeed, the removal of such regulatory cells, e.g., by neonatal thymectomy, is thought to be a major mechanism by which disease can develop. The culprit T helper type 1 (Th1) CD4+ T cells recognize either the alpha- or beta-subunits of the gastric H+/K+ ATPase, but the beta-subunit appears to be the initiating autoantigen, while the alpha-subunit may have a role in perpetuating disease. Since no specific environmental modifiers are identifiable, the origins of the disease are intrinsic; this is illustrated by the capacity of a cytokine (GM-CSF)-dependent inflammatory stimulus in the stomach to initiate EAG, according to a transgenic model in which thymectomy is dispensible. Thus, EAG is an exquisite model for a reductionist analysis of the multiple elements that in combination induce autoimmunity in humans.     

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

The aim of this study was to identify immunoreactive domains on human ribosomal P0, P1 and P2 proteins, other than the C-22 peptide, to develop a novel ELISA using a combination of these proteins and to compare this ELISA with one using the C-22 peptide. Human recombinant P0, P1, P2 and mutant P0 lacking the homologous C-22 peptide (N-P0) were produced in bacteria and tested by ELISA and immunoblotting using sera from 48 patients with systemic lupus erythematosus (SLE), 48 with an unrelated inflammatory disorder (Crohn's disease) and 47 healthy controls. ELISA with P0, P1 and P2, premixed at equimolar concentrations, gave higher OD readings than each protein tested individually. Eighteen SLE sera tested positive by ELISA with premixed P0, P1, P2 but only 3 tested positive with the C-22 peptide. Twenty-two SLE sera reacted positively, as determined by immunoblotting, with 5 different P protein combinations: P1P2, P0P1P2, P1, P0P1, P0 and P1. Only sera reactive with all three P proteins reacted with the C-22 peptide, with absent or minimal reactivity with N-P0. Native antigens yielded sensitivity (6/48, 13%) similar to the C-22 peptide assay. An ELISA with premixed P1 and P2 gave higher OD values than the arithmetic means with P1 or P2. Fifteen SLE patients had antibodies to double stranded (ds)-DNA, of which 6 also had antibodies to P0P1P2 by ELISA but 12 reactive with P0P1P2 did not have discernable ds-DNA antibodies. Ribosomal P autoantibodies react mainly with epitopes N-terminal to a homologous C-22 peptide. An ELISA with premixed P0, P1 and P2 has 5-fold greater sensitivity (38%) for SLE than an assay with the conventional C-22 peptide (7%). The combined sensitivity for SLE for antibodies to P0P1P2 and ds-DNA is 56%, higher than C-22 and ds-DNA, 38%. Only one of the SLE patients had neuropsychiatric lupus.     

Parietal cell surface reactive autoantibody in pernicious anaemia demonstrated by indirect membrane immunofluorescence.

We examined, in a 'double blind' study, 60 sera from patients with pernicious anaemia for immunofluorescence reactivity with the surface membranes of viable parietal cells isolated from dog stomachs. Fifty-three sera (88%) gave an IgG autoantibody reaction with the surface membranes of parietal cells. Surface staining was also seen with parietal cells from monkey, pig, rat and mouse. The parietal cell surface reactive autoantibody was not found in any of 14 sera from patients with chronic active hepatitis, 10 from patients with systemic lupus erythematosus and 50 from healthy persons. The surface reactivity autoantibody was present in 13 of 14 sera without parietal cell microsomal antibody, 28 of 31 sera without intrinsic factor antibody and in four of four sera without microsomal and intrinsic factor antibodies. Absorption with parietal cell enriched gastric mucosal cells neutralized the activity of the surface reactive but not the microsomal antibody and cross absorption with gastric microsomes neutralized the activity of the microsomal but not the surface reactive antibody. Surface staining of parietal cells was not abolished by absorption with dog or rat hepatocytes, dog or rat kidney cells, human fibroblasts or human AB red blood cells. The results suggest that the parietal cell surface reactive antibody is probably different from the microsomal antibody. Immune reactions of the cell surface reactive antibody with parietal cell surface antigens may play a role in the pathogenesis of the gastric lesion in pernicious anaemia.     

Immunization with gastric H+/K(+)-ATPase induces a reversible autoimmune gastritis.

The gastric H+/K(+)-ATPase has been implicated as a major autoantigen in pernicious anaemia in humans and in thymectomy-induced autoimmune gastritis in mice. Here we have shown that autoimmune gastritis can be generated by direct immunization of non-thymectomized BALB/c mice with mouse gastric H+/K(+)-ATPase in complete Freund's adjuvant. The gastritis was characterized by infiltration of the gastric submucosa and mucosa with macrophages, CD4+ and CD8+ T cells, and B cells and by circulating autoantibodies to the H+/K(+)-ATPase. The mononuclear infiltrate within the gastric mucosa was accompanied by loss of parietal and zymogenic cells and accumulation of small immature epithelial cells. Splenocytes from gastritic mice adoptively transferred gastritis to naive recipients. Cessation of immunization resulted in decrease in autoantibody titre and regeneration of parietal and zymogenic cells. The results directly confirm that the gastric H+/K(+)-ATPase is the causative autoantigen in the genesis of autoimmune gastritis. Recovery of the lesion following cessation of immunization suggests that homeostatic mechanisms can reverse a destructive autoimmune process.