CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence

It is known that replicative senescence of endothelium in vivo contributes at least partially to age-related vascular disorders such as arteriosclerosis. However, the genes involved in this process remain to be identified. In this study, we employed a proteomics-based approach to identify candidate genes using in vitro cultured human umbilical vein endothelial cells (HUVECs) as an experimental model for replicative senescence. By comparing protein spots from young and senescent HUVECs using two-dimensional electrophoresis, we identified three up-regulated proteins and five down-regulated proteins in senescent HUVECs as compared to young HUVECs, whose alteration was not observed during replicative senescence of primary human fibroblasts. Consistent results were obtained in Western blotting analysis using specific antibodies raised against some of these proteins, whereas there were no significant changes in the mRNA levels of these genes during senescence of HUVECs. Among them, cathepsin B, a protease participating in both intracellular proteolysis and extracellular matrix remodeling was observed to be dramatically up-regulated in senescent HUVECs and whose activity is known to be up-regulated in atherosclerotic lesions with senescence-associated phenotypes in vivo. Additional proteins, including cytoskeletal proteins and proteins involved in the processes of synthesis, turnover and modification of protein, were identified, whose function in endothelium was previously unsuspected. These proteins identified by a proteomics-based approach using cultured HUVECs may be involved not only in replicative senescence but also in functional alterations in vascular endothelial cells with senescence-associated phenotypes and may serve as molecular markers for these processes.     

Nitrate respiratory metabolism in an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6

Hydrogenobacter thermophilus strain TK-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. Nitrite was detected as the product of a respiratory reaction. 15NO, 15N2O, and 15N2 were detected with Na15NO3 as an electron acceptor. Western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd1-type nitrite reductase from Pseudomonas aeruginosa. The positive bands, which had molecular masses similar to that of the heme cd1-type nitrite reductase, were also stained by heme staining. These results indicate that nitrite reductase of strain TK-6 is a heme cd1-type enzyme. Activity of ATP:citrate lyase, one of the key enzymes of the reductive TCA cycle, was detected in cell-free extract of cells cultivated on nitrate, which indicates that the cycle operates during anaerobic growth.     

A method for preparing 2- to 50-µm-thick fresh-frozen sections of large samples and undecalcified hard tissues

This article describes a method for preparing 2- to 50-micron-thick fresh-frozen sections from large samples and completely calcified tissue samples. In order to perform the more routine work involved, a tungsten carbide disposable blade was installed to a heavy-duty sledge cryomicrotome. An entire 10-day-old rat and bone and tooth samples from a 7-month-old rat were rapidly frozen. The frozen samples were attached to the cryomicrotome stage. The cutting surface of the samples was covered with a polyvinylidene chloride film coated with synthetic rubber cement and cut at -25 degrees C. The soft tissues and the hard tissues were satisfactorily preserved and all tissue cells were easily identifiable. Enzymatic activity in the fresh sections was much stronger than that in chemically fixed and/or decalcified sections. The sections permitted histological and histochemical studies without trouble. In addition, the sections can be used for multiple experiments such as immunohistochemistry, in situ hybridization, and electron microprobe X-ray micro-analysis. This method can be used with conventional cryomicrotome equipment.     

Effect of a New Variety of Apis mellifera Propolis onMutans Streptococci

The effects of a new variety of propolis, from Northeastern Brazil (BA), on growth of mutans streptococci, cell adherence, and water-insoluble glucan (WIG) synthesis were evaluated. Propolis from Southeastern (MG) and Southern (RS) Brazil were also tested as an extension of our previous work. Ethanolic extracts of propolis (EEP) were prepared and analyzed by reversed-phase HPLC. For the antibacterial activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEPs against Streptococcus mutans, S. sobrinus, and S. cricetus were determined. Cell adherence of S. mutans and S. sobrinus to a glass surface was measured spectrophotometrically at 550 nm. WIG synthesized from sucrose by glucosyltransferase (Gtf) was extracted and quantified by the phenol-sulfuric method. The HPLC profile of the new variety of propolis was entirely different from Southeastern and Southern propolis. Neither flavonoid aglycones nor p-coumaric acid were detected in EEP BA. All EEPs demonstrated biological activities against mutans streptococci; EEP BA showed the highest potency in all in vitro parameters evaluated in this study. The ranges of MIC values were 50 (EEP BA)–400 μg/ml (MG), for S. mutans; and 25 (BA)–400 μg/ml (MG), for S. sobrinus and S. cricetus. The bactericidal concentration of EEPs was four to eight times the MIC values. The adherence of S. mutans and S. sobrinus cells and WIG synthesis were markedly inhibited by EEPs, demonstrating significant inhibition at all concentrations compared with the control (80% ethanol) (p < 0.05). EEP BA showed 80% inhibition of cell adherence and WIG synthesis at concentrations as low as 12.5 and 7.8 μg/ml, respectively. The results show that the new variety of propolis was exceptionally effective in all in vitro parameters tested against mutans streptococci; biological effects of propolis are likely not to be due solely to flavonoids and (hydroxy)cinnamic acid derivatives. Received: 14 February 2000 / Accepted: 8 May 2000