Primary and secondary structure of bovine retinal S antigen (48-kDa protein).

The complete amino acid sequence of bovine S antigen (48-kDa protein) has been determined by cDNA and partial amino acid sequencing. A 1623-base-pair (bp) cDNA contains an open reading frame coding for a protein of 404 amino acids (45,275 Da). Tryptic peptides and cyanogen bromide peptides of native bovine S antigen were purified and partially sequenced. All of these peptides were accounted for in the long open reading frame. Searching of the National Biomedical Research Foundation data bank revealed no extensive sequence homology between S antigen and other proteins. However, there are local regions of sequence similarity with alpha transducin, including the sites subject to ADP-ribosylation by Bordetella pertussis and cholera toxins and the phosphoryl binding-sites. Secondary structure prediction and circular dichroic spectroscopy show that S antigen is composed predominantly of beta-sheet conformation. Acid-catalyzed methanolysis suggests the presence of low levels of carbohydrate in the molecule.     

Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases.

The fibronectin (FN) receptor in avian cells has been characterized previously as a complex of three membrane glycoproteins of about Mr 160,000, Mr 140,000, and Mr 120,000 (simply termed protein band 1, band 2, and band 3, respectively). Monoclonal antibodies to the band 3 protein of the complex prevent FN and laminin binding both in vivo and in vitro and enable the detection of the receptor proteins in the plasma membrane and in adhesion plaques. Association of the FN receptor proteins with the adhesion-plaque protein talin also has been reported. We now find that the band 2 and band 3 proteins in the complex are phosphorylated in Rous sarcoma virus-transformed chicken cells but not in normal chicken cells. Phosphorylation occurs predominantly on tyrosine and is accompanied by a reorganization of the receptor complex in the membrane of the transformed cells. Whereas normal cells contain the FN receptor in focal contacts and cellular processes between cells, v-src-transformed cells exhibit a more diffuse distribution of this receptor. In addition to the viral v-src oncogene, cells transformed by other avian oncogenes that also encode tyrosine kinases (v-fps, v-erbB, and v-yes) also express the receptor complex proteins in the phosphorylated state regardless of whether the transforming protein is detectable in adhesion plaques. These results suggest that the altered FN and laminin receptor proteins may contribute to the transformed phenotype, but their significance and role in the transformed state remain to be established.     

Trends in 30-Day Readmission Rates for Medicare and Non-Medicare Patients in the Era of the Affordable Care Act

Background

Temporal changes in the readmission rates for patient groups and conditions that were not directly under the purview of the Hospital Readmissions Reduction Program (HRRP) can help assess whether efforts to lower readmissions extended beyond targeted patients and conditions.

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.     

Myocardial sulfhydryl pool alterations occur during reperfusion after brief and prolonged myocardial ischemia in vivo

Myocardial sulfhydryl (SH)-containing compounds, including reduced glutathione (GSH), are both defenses against and potential markers of reactive oxygen metabolite injury during ischemia and reperfusion. We examined the alterations in GSH and other myocardial SH pools during reperfusion in anesthetized dogs exposed to brief (15 minutes, n = 7) or prolonged (90 minutes, n = 6) regional ischemia caused by occlusion of the left anterior descending artery. Ninety minutes of ischemia followed by 5 hours of reperfusion, which resulted in myocardial necrosis of 43.9 +/- 4.0% of the area at risk, caused a 22% reduction in total myocardial SH groups (p less than 0.01), a 57% decrease in nonprotein myocardial SH groups (p less than 0.01), a 56% decrease in GSH (p less than 0.01), and a 62% decrease in non-GSH, nonprotein SH groups (p less than 0.02). However, protein SH groups were not significantly reduced (12% decrease, p = NS). Also, myocardial release of GSH and oxidized glutathione (GSSG) into the coronary venous effluent occurred during early reperfusion. In contrast, 15 minutes of ischemia, followed by 30 minutes of reperfusion, did not alter myocardial total SH groups, protein SH groups, or GSH (9% decrease, p = NS); nor was there reperfusion release of GSH or GSSG. However, even with brief ischemia, nonprotein SH groups decreased 23% (p less than 0.05), due mainly to a 59% decrease in the non-GSH, nonprotein SH pool (p less than 0.05). These changes after brief ischemia occurred without alterations in myocardial GSSG or the GSH/GSSG ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4- hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.     

Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.