Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis.

BACKGROUND: Calcific aortic stenosis (AS), the most frequent heart valve disorder in developed countries, leads to the calcification and fibrous thickening of the valve. While several studies have addressed the process of valvular calcification, the molecular pathomechanisms of the extensive matrix remodeling remain unclear. Because inflammation is present in stenotic valves, we hypothesized that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) might influence cell proliferation and regulate the expression and activation of matrix metalloproteinases (MMPs)--enzymes that are thought to be involved in calcific AS. METHODS: Immunohistochemistry for leukocytes, TNFalpha, MMP-1, and the endogenous MMP inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 was performed on human stenotic (n = 19) and control (n = 8) valves. Primary cultures of human aortic valve myofibroblasts were incubated with and without TNFalpha, and cell proliferation was assessed. The expression and activation of MMP-1 were detected by Western blotting and a specific MMP-1 activity assay. RESULTS: Control valves showed scattered macrophages and low expression of TNFalpha, MMP-1, and TIMP-1. In stenotic valves, leukocyte infiltration and a strong, colocalized expression of TNFalpha and MMP-1 were present, while TIMP-1 remained unchanged. Double-label immunofluorescence localized TNFalpha mainly to macrophages. In cultured human aortic valve myofibroblasts, TNFalpha stimulated proliferation and induced a time-dependent increase in MMP-1 expression and activation, while TIMP-1 remained unchanged. CONCLUSION: The results indicate that matrix remodeling in calcific AS involves the expression and activation of MMPs. Activated leukocytes, by the secretion of TNFalpha, may stimulate valvular myofibroblasts to proliferate and express MMPs, thus regulating actively the matrix remodeling in calcific AS.     

New copolymethacrylates with azo-chromophores in the side chain

The synthesis and characterization of new poly({6-[4-(4-cyanophenylcarbamoyl)phenoxy]hexyl methacrylate}-co-{6-[4-(4- cyanophenylazo)phenoxy]hexyl methacrylate}) are reported. Their liquid-crystalline properties are investigated using differential scanning calorimetry, polarizing microscopy and X-ray diffraction techniques. The glass transition temperatures and the clearing points can be influenced by variation of the copolymer composition. The substances offer a relatively broad temperature range of mesomorphic properties suitable for photochemical studies.     

Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.     

Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors A comparison between velocity sedimentation and discontinuous density gradients

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 × 10⁸. The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.