Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [35S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

Osteoporotic hip fracture is a major public health issue. Estimation of the outcome and maximization of functional recovery after fracture is very important in the treatment of older patients. The purposes of this study were to clarify the functional outcomes after the treatment of hip fracture and to identify the factors that influence functional recovery. In the present study, 228 patients admitted to an acute-care hospital from January 2016 to June 2018 were evaluated. The patients were categorized into a trochanteric fracture group (n = 128) and a neck fracture group (n = 100). We retrospectively reviewed their ambulation ability 6 months after fracture using the Functional Ambulation Category (FAC) score. The other survey items were the presurgical duration, length of hospital stay, time until beginning to walk using parallel bars, complications affecting treatment, and mortality rate. The 6-month follow-up rate was 54.4% (n = 124). The results showed that the patients with trochanteric fracture were significantly older than those with neck fracture (86 vs. 82 years, respectively; p = 0.03). In total, 85.0% of patients with trochanteric fracture and 92.2% of patients with neck fracture were independent ambulators before injury (FAC score of 4 or 5). The FAC score 6 months after fracture was positively correlated with the FAC score before fracture and at discharge (all p<0.001) and negatively correlated with patient age (p<0.001) and presurgical duration for patients with neck fracture (p = 0.04). There was no statistically significant correlation with the length of hospital stay or the time until beginning to walk using parallel bars. In conclusion, patients with trochanteric fractures were older than those with neck fractures. In both fracture types, walking recovery 6 months after hip fracture was related to the FAC score before injury and at discharge from an acute-care hospital but not to the time until beginning to walk using parallel bars.

We have previously reported that a non-receptor-type protein-tyrosine kinase p7PYk, exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p7PYk, we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p7Pyk was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p7Pyk rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p7Pyk in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p7Pyk significantly increased approximately tenfold and reached a maximum at 10 s ; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p7PYk with phosphatidylinositol 3-kinase and tyrosine phosphorylation of ~7 2 '~~ were observed. These results suggest that translocation, activation, and association of p72'yk with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events.Blood platelets, which are terminally differentiated nonproliferative cells devoid of growth or the capability cell division, contain exceptionally high levels of protein-tyrosine kinase activity in both particulate and cytosolic fractions [l-31. Several studies have recently established that various agonists such as thrombin, collagen, platelet-activating factor, and vasopressin, stimulate protein-tyrosine phosphorylation in platelets [4-61. These observations suggested that proteintyrosine kinase may regulate certain cellular processes in platelets that are distinct from cell growth.We previously reported a porcine gene encoding a nonreceptor-type 72-kDa protein-tyrosine kinase (~7 2 "~) which has unique structural characteristics, possessing the second src homology region 2 (SH2) domain instead of SH3 in its amino-acid sequence [7]. The expression of p7PYk has been demonstrated in porcine splenocytes, platelets, peripheral blood lymphocytes, polymorphonuclear leukocytes and tonsilocytes [8, 91. Recently, we showed that ~7 2 " ' was observed in both particulate and cytosolic fractions of platelets and was rapidly activated following thrombin stimulation [ 8,Correspondence to H. Yamamura, Department of Biochemistry, Fax: +81 776 61 8102. Abbreviations. p72"yk, 72-kDa non-receptor-type protein-tyrosine kinase; SH, src homology region.Enzymes. Protein-tyrosine kinase (EC 2.7

The function and the outside-in signaling pathways of IIbβ3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of IIbβ3. Binding of soluble fibrinogen to the cells via IIbβ3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by IIbβ3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin β3 and a complex formation of integrin β3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways. © 1998 by The American Society of Hematology.