The integrin ␣ 9 ␤ 1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH 2 -terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for ␣ 9 ␤ 1 . In this report, we used site-directed mutagenesis and synthetic peptides to identify the ␣ 9 ␤ 1 recognition sequence in osteopontin. ␣ 9 -transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH 2 -terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-␣ 9 monoclonal antibody Y9A2, indicating the presence of a non-RGD ␣ 9 ␤ 1 recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on ␣ 9 ␤ 1 -mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr 165 , blocked ␣ 9 ␤ 1 -mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin ␤ 1 subunit on ␣ 9 -transfected, but not on mocktransfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to ␣ 9 ␤ 1 and is responsible for ␣ 9 ␤ 1 -mediated cell adhesion to the NH 2 -terminal fragment of osteopontin.Integrins are cell surface heterodimeric receptors that mediate cell-cell and cell-extracellular matrix adhesion (1, 2). Upon ligation by a wide variety of ligands, integrins can initiate signaling cascades that regulate cell growth, cell death, migration, polarization, and tissue remodeling (3). Integrins recognize a surprisingly large number of functionally diverse proteins as ligands, and the list of known integrin ligands continues to grow. New integrin ligands have been identified, and drugs targeting integrins have been developed as a consequence of the description of short linear amino acid sequences that directly bind to integrins. For example, the integrins, and ␣ v ␤ 8 bind to sequences containing the tri-peptide sequence Arg-Gly-Asp (RGD). Several new and biologically important integrin ligands have been identified based on the presence of this sequence (4, 5). Drugs modeled on the structure of the RGD sequence are being used or tested to inhibit integrin function for treatment of thrombosis, inflammation, atherosclerosis, osteoporosis, and cancer (5). The RGD sequence has also been exploited to target cell surface integrins to enhance gene delivery (6). We have previously identified the recognition sequence for the integrin ␣ 9 ␤ 1 in tenascin-C and found that this sequence did not include RGD, but was homologous to the ␣ 4 ␤ 1 recognition sequence in the inducible endothelial adhesion molecule VCAM-1 (7). This finding led to our identification of ␣ 9 ␤ 1 as a receptor for VCAM-1 (8).Osteopontin is a phosphorylated acidic gly...

BACKGROUND.Tissue factor (TF), a cell surface receptor of factor VIIIVIIa, was

NOTESymbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE-cellulose column and assayed for protein phosphatases for tau factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form 2 (minor), were separated. Each peak was in a single form on Sephacryl S-300 column chromatography. Both peaks 1 and 2 dephosphorylated tau factor phosphorylated by Ca2+/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The Km values were in the range of 0.42-0.84 microM for tau factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+ and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40-60 microM. These results suggest that dephosphorylation of tau factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of tau factor.

Introduction. Gemcitabine and cisplatin (GC) is a gold-standard first-line systemic chemotherapy for advanced urothelial carcinoma (UC). However, it may cause severe adverse effects such as renal toxicity, gastrointestinal toxicity, and neurotoxicity. Sarcopenia is the age-related loss of skeletal muscle mass. A correlation between sarcopenia and the oncological prognosis has been reported. In UC, several studies have noted that patients with sarcopenia had a greater incidence of complications and worse survival after radical cystectomy or chemotherapy. Our institute introduced gemcitabine and nedaplatin (GN) for UC patients with renal failure. We investigated whether the presence of sarcopenia predicted the prognosis of patients with advanced UC who were treated by GN chemotherapy. Methods. A total of 27 patients (male, n = 21; female, n = 6) received GN therapy for metastatic UC from 2005 to 2016. The institutional review board of Yokohama City University Hospital approved this study. The psoas muscle index (PMI, cm2/m2) was calculated using this formula: right psoas muscle area (cm2)/the square of the body height (m2). The overall survival (OS) of the high PMI group (male: ≥2.49, female: ≥2.07) and low PMI group (male: <2.49, female: <2.07) was compared. Results. Kaplan-Meier survival curves and a log-rank test revealed that the high PMI group had significantly better OS than the low PMI group (p = 0.015). The mean survival of the high and low PMI groups was 561 days and 223 days, respectively. Conclusions. In the present study, we revealed that sarcopenia (a low psoas muscle volume) might be a predictive factor for poorer overall survival in patients with advanced urothelial carcinoma who are undergoing GN chemotherapy.

Hyperphosphatemia is an important determinant of morbidity and mortality in patients with chronic kidney disease (CKD). Patients with CKD are advised to consume a low phosphate diet and are often prescribed phosphate-lowering drug therapy. However, commercially processed food and drinks often contain phosphate compounds, but the phosphate level is not usually provided in the ingredient list, which makes it difficult for CKD patients to choose a correct diet. We conducted a survey of the awareness of food/beverages containing artificially added phosphate among CKD patients undergoing hemodialysis. The subjects were 153 patients (77 males and 76 females; average age 56±11 years) who were randomly selected from the Dialysis Center of Hirosaki City, Japan. The subjects were provided with a list of questions. The survey results showed that 93% of the subjects were aware of the presence of high sugar content in soda, whereas only 25% were aware of the presence of phosphate (phosphoric acid) in such drinks. Despite 78% of the subjects being aware of the detrimental effects of consumption of a high phosphate diet, 43% drank at least 1 to 5 cans of soda per week and about 17% consumed “fast food” once each week. We also assessed the immediate effects of high-phosphate containing carbonated soda consumption by determining urinary calcium, phosphate, protein and sugar contents in overnight fasted healthy volunteers (n = 55; average age 20.7±0.3 years old, 20 males and 35 females). Significantly higher urinary calcium (adjusted using urinary creatinine) excretion was found 2 h after consuming 350 ml of carbonated soda compared to the fasting baseline level (0.15±0.01 vs. 0.09±0.01, p = 0.001). Our survey results suggest that CKD patients undergoing hemodialysis are not adequately aware of the hidden source of phosphate in their diet, and emphasize the need for educational initiatives to raise awareness of this issue among CKD patients.

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