Citations

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by α- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

Background— Clinical and radiologic predictors of cerebral infarction occurrence and location after aneurysmal subarachnoid hemorrhage have been seldom studied. Methods— We evaluated all patients admitted to our hospital with aneurysmal subarachnoid hemorrhage between 1998 and 2000. Cerebral infarction was defined as a new hypodensity located in a vascular distribution on computed tomography (CT) scan. Results— Fifty-seven of 143 patients (40%) developed a cerebral infarction. On univariate analysis, occurrence of cerebral infarction was associated with a worse World Federation of Neurological Surgeons grade ( P =0.01), use of ventriculostomy catheter ( P =0.01), preoperative vasospasm ( P =0.03), surgical clipping ( P =0.02), symptomatic vasospasm ( P <0.01), and vasospasm on transcranial Doppler ultrasonography (TCD) or repeat angiogram ( P <0.01). On multivariable analysis, only presence of symptoms ascribed to vasospasm ( P <0.01) and evidence of vasospasm on TCD or angiogram predicted cerebral infarction ( P <0.01). TCD and angiogram agreed on the diagnosis of vasospasm in 73% of cases (95% CI, 63% to 81%), but the diagnostic accuracy of this combination of tests was suboptimal for the prediction of cerebral infarction occurrence (sensitivity, 0.72; specificity, 0.68; positive predictive value, 0.67; negative predictive value, 0.72). Location of the cerebral infarction on delayed CT was predicted by neurological symptoms in 74%, by aneurysm location in 77%, and by angiographic vasospasm in 67%. Conclusions— Evidence of vasospasm on TCD and angiogram is predictive of cerebral infarction on CT scan but sensitivity and specificity are suboptimal. Cerebral infarction location cannot be predicted in one quarter to one third of patients by any of the studied clinical or radiological variables.

Abstract Background: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. Methods: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 μL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13 761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. Results: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. Conclusions: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala.

Acute ulcers of the gastric mucosa are found in a large percentage of dogs and cats dying after adrenalectomy. These ulcers seem to develop during the moribund period. They are apparently peptic ulcers forming at the site of local hemorrhages in the gastric mucosa. They are true acute ulcers, usually penetrating to the muscularis mucosa with a total loss of epithelium. They develop in the absence of pancreatic secretion and bile. However, they appear to develop only in an acid medium.

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.

Although interleukin 1 (IL-1) receptor signaling events in T helper lymphocytes are incompletely characterized, events associated with translocation of the transcription factor NF-kappa B are receptor-proximal assays of ligand-initiated responses. In this report we demonstrate that the transient nature of IL-1-induced NF-kappa B nuclear translocation occurs as a consequence of ligand-induced receptor desensitization. Other receptor-initiated events including induction of I kappa B alpha phosphorylation, expression of c-jun and junB mRNA, and costimulatory effects on IL-2 synthesis also are altered by IL-1 receptor desensitization. IL-1 receptor desensitization is not initiated by tumor necrosis factor, which also stimulates NF-kappa B translocation, and is not a consequence of alterations in either IL-1 receptor expression or binding affinity. In the absence of IL-1, the effects of desensitization are completely reversed within 18 h. Since IL-1 desensitization is initiated under conditions of low receptor occupancy, it is likely that receptor desensitization results from alterations to a receptor-proximal transducer, rather than from direct modification of the IL-1 receptor. These results suggest that the cyclic nature of the events in the T helper lymphocyte activation program can be controlled, in part, by the reversible desensitization of cell surface IL-1 receptors.

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3-kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor-deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3-kinase, in IL-2-stimulated T cells.

Giant cell arteritis (GCA) is a spontaneous vasculitic syndrome that specifically targets the walls of medium and large arteries. Vascular lesions are characterized by patchy granulomatous infiltrates composed of T cells, macrophages, histiocytes, and giant cells. To test the hypothesis that a locally residing antigen recruits T cells into the vessel walls, we have analyzed T cell receptor (TCR) molecules of tissue infiltrating T cells. A total of 638 CD4+ T cell clones were isolated from temporal artery specimens of three patients with GCA. Analysis of TCR molecules for the usage of V beta 1-V beta 20 revealed that all TCR V beta elements were represented, demonstrating that interleukin 2 (IL-2)-responsive T cells infiltrating the tissue are highly diverse. To detect expanded T cell specificities, we made use of the patchy character of the inflammatory disease and compared the TCR repertoire of T cells established from independent vasculitic foci of the same artery. Sequence analysis of TCR V beta chains documented that individual TCR specificities were present in multiple copies, indicating clonal expansion. T cells with identical beta chains were isolated from distinct inflammatory foci of the same patient. These specificities represented only a small fraction of tissue-infiltrating T cells and involved the V beta 5.3 gene segment in the two patients sharing the HLA-DRB1*0401 allele. The third complementarity determining region of clonally expanded TCR beta chains was characterized by a cluster of negatively and positively charged residues, suggesting that the juxtaposed antigenic peptide is charged. The sharing of identical T cell specificities by distinct and independent regions of the granulomatous inflammation suggests that these T cells are disease relevant and that their repertoire is strongly restricted. These data suggest that an antigen residing in the arterial wall is recognized by a small fraction of CD4+ T cells in the inflammatory process characteristic for GCA.