Circular dichroism of ketoprofen complexed to serum albumins: Conformational selection by the protein: A novel optical purity determination technique

Complexation of 2-(3′-benzoylphenyl)propionic acid (ketoprofen), 1 , to bovine serum albumin (BSA) results in an intense negative circular dichroism in the ketonic n → π* band of the benzoylphenyl moiety. This high CD contrasts with the weak CD of 1 -enantiomers dissolved in common solvents. Furthermore, a number of chiral and achiral molecules containing the benzophenone moiety are easily complexed to BSA: all these complexes show an intense CD at the same transition. To account for the observed CD intensities of the above molecules, it appears that BSA complexation markedly shifts the equilibrium between strongly asymmetric, antipodic conformers. Dissymmetry of these conformers is connected to the instability of a structure with phenyl rings coplanar to the carbonyl chromophore, as also indicated by molecular mechanics calculations. The magnification of the Cotton effects of the 1 -antipodes, due to the protein, can be used to measure the optical purity of 1 -samples with excellent precision. In contrast with BSA, human SA is unable to recognize the chirality of 1 -antipodes; oleic acid cocomplexation modifies this fact as well as other features of the binding. © 1995 Wiley-Liss, Inc.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells

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Genetic regulation of MUC1 expression by Helicobacter pylori in gastric cancer cells

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.     

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris–HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65 °C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.     

A rare fraction of human hematopoietic stem cells with large telomeres

The lack of specific markers for stem cells makes the physical identification of this compartment difficult. Hematopoietic stem cells differ in their repopulating and self-renewal potential. Our study shows that multiple classes of human hematopoietic CD34+ greatly differ in telomere length. Flow-cytometry-based fluorescent in situ hybridization and confocal microscopy of CD34+ cells has revealed remarkable telomere length heterogeneity, with a hybridization pattern consistent with different classes of human hematopoietic progenitor cells. These results also point to the existence of a significant clonal heterogeneity among primitive hematopoietic cells and provide the first evidence of a rare fraction of CD34+ cells with large telomeres in humans. Marta García-Escarp and Vanessa Martinez-Muñoz contributed equally to this work.This work was supported by a grant to J.P. from the Spanish Ministry of Science and Technology (SAF2002-02618) and by a grant to V.M.-M. from DakoCytomation.     

Expression of CD147 on phorbol-12-myris-tate-13-acetate (PMA)-treated U937 cells differentiating into foam cells

Monocytes, macrophages, and foam cells expressing CD147 can stimulate the production of several matrix metalloproteinases (MMPs) associated with the development of atherosclerosis. We defined the CD147 expression profile and examined the correlation between foam cell development and MMP-2, -9 expressions. Foam cells were derived from U937-stimulated macrophages using various concentrations of oxidized low-density lipoprotein (ox-LDL). PMA-stimulated U937 cells had a 4- to 5-fold increase in CD147 mRNA compared to undifferentiated monocytes and membrane-associated (mCD147) on foam cells decreased in response to ox-LDL in a dose-dependent manner compared to untreated macrophages. In contrast, ox-LDL treatment increased the levels of soluble CD147 (sCD147) and MMP-2, -9 in a dose-dependent manner. Our data suggested that monocyte differentiation up-regulated CD147 expression and lipid enrichment of foam cells had no effect on CD147 mRNA expression. Lipid loading in macrophages reduced mCD147 expression while increasing the levels of MMP-2, -9 and sCD147 in supernatants.     

CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9⁺ cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.     

Downregulation of CD2-associated protein impaired the physiological functions of podocytes

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.