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Biomedical subjects

M M Burger

Publications and source records attributed to M M Burger.

At least 19 recordsLinked to original sources
PubMed2003

Circular proteoglycans from sponges: first members of the spongican family.

Species-specific cell adhesion in marine sponges is mediated by a new family of modular proteoglycans whose general supramolecular structure resembles that of hyalectans. However, neither their protein nor their glycan moieties have significant sequence homology to other proteoglycans, despite having protein subunits equivalent to link proteins and to proteoglycan monomer core proteins, and glycan subunits equivalent to hyaluronan and to the glycosaminoglycans of hyalectans. In some species, these molecular components are assembled into a structure with a circular core formed by the link protein- and hyaluronan-like subunits. Besides their involvement in cell adhesion, these sponge proteoglycans, for which we propose the term spongicans, participate in signal transduction processes and are suspected to play a role in sponge self-nonself recognition. Their in vivo roles and the mild methods used to purify large amounts of functionally active spongicans make them ideal models to study the functions and possible new applications of proteoglycans in biomedical research.

Amino Acid Sequence↗
PubMed2001

Molecular interaction between human tumor marker protein p150, the largest subunit of eIF3, and intermediate filament protein K7.

The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180). Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas. The function of p150 in cancer and initiation of translation are not well understood. Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein. The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro. The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835. The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150. By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150. Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell.

Binding Sites↗
PubMed2001

Mechanisms regulating c-met overexpression in liver-metastatic B16-LS9 melanoma cells.

Liver selected B16-LS9 melanoma cells show a dramatic overexpression of the proto-oncogene c-met, the cellular receptor for hepatocyte growth factor/scatter factor. As a consequence, c-met becomes constitutively active, and the cells become more responsive to hepatocyte growth factor stimulation. We have investigated the molecular mechanisms regulating c-met expression in both the parental line B16-F1, which has low expression levels, and the liver-specific B16-LS9, overexpressing c-met. Overexpression is observed at the protein and mRNA levels, however without further evidence of gene amplification or rearrangement. c-met promoter activity was higher in B16-LS9 than B16-F1 cells, and also a nuclear run-off showed higher transcription levels in B16-LS9 cells. Moreover, we found that c-met mRNA had a longer half-life in B16-LS9 cells, thus indicating also the involvement of post-transcriptional regulation mechanisms. Finally, we found evidence that autonomous activation of the melanocortin receptor-1 (MCR-1) is at least partially responsible for c-met upregulation in B16-LS9 cells, since treatment of the cells with a potent MSH antagonist (the agouti peptide) has strong down-regulatory effects.

Animals↗
PubMed2001

[Mechanisms of metastasis].

Until a primary tumor can successfully produce clinically relevant metastases, the seed cells have to go through a series of stepwise changes (mutations) which have to overcome a sizeable cascade of obstacles. Some of the experimentally proven and clinically suspected changes involve cell surface adhesion, general and local growth factor responsiveness, protease and glycosidase production, motility and deformability. In addition to this tumor cell centered view, one begins to broaden the vision to include changes in the interactions between the tumor cell and its immediate environment. In this context it turns out that the tumor cell seems to learn how to entice neighboring cells and tissues to support its progress with several different responses: extracellular matrix degrading enzymes, growth factors in the target organ, angiogenesis etc. The multitude of phenotype variants is due to genome instability, which is higher in metastatic cells than in primary tumor cells. Recently, it could be shown that even chemotherapy drug resistance is increased in the metastatic organ surrounding (lung) compared to non-metastatic organ targets. The role of the immune system for organ specific metastasis is--if anything--less clear than earlier assumed. Important for the surgeon is the fact that his efforts with the primary tumor could become yet even more successful if the dormant or micrometastases could be abolished. New therapeutic approaches include specific inhibitors of angiogenesis receptors (tyrosine kinase inhibitors). Recently, it was found that continuous low dose chemotherapy together with antiinflammatory agents which block endothelial cell migration and growth prevent not only tumor growth, but abolish micrometastases efficiently in animals. Clinical phase II studies are underway. This, as so many other promising starts in cancer therapy, needs, however, solid confirmation on a very expanded scale before any hopes can be raised.

Animals↗
PubMed2000

Structure and function of the C-terminal hypervariable region of K-Ras4B in plasma membrane targetting and transformation.

The C-terminal hypervariable domain of K-Ras4B targets the protein to the plasma membrane by a combination of positive charge and a hydrophobic signal (farnesyl group). We analysed the contribution of several structural features of the domain: net charge, charge distribution, amino acid sequence and lipid specificity to membrane targetting and function by using artificial 'hypervariable' domains fused to either EGFP or V12KRas4B. We found that charge and a lipid residue are sufficient for plasma membrane localization and function of the constitutively active V12K-Ras4B. However, the amount of net charge, charge distribution and the length of the anchoring domain are important. Increasing the net charge and concentrating it close to the C-terminus increases not only the percentage of membrane bound protein, but also shifts the distribution from internal membranes, including the nuclear envelope, to the plasma membrane. While plasma membrane binding is necessary for V12K-Ras4B activity (MAPK activation and focus formation), we found that there are additional restrictions. In particular, mutants with very highly charged domains that bind almost exclusively to the plasma membrane show less transforming potential than expected. In addition, a construct with a short 'hypervariable' domain (7 amino acids) also has decreased transformation activity. These results suggest that specific interactions between K-Ras4B and the plasma membrane are required.

3T3 Cells↗
PubMed2000

Supramolecular structure of a new family of circular proteoglycans mediating cell adhesion in sponges.

Aggregationfactors are the molecules responsible for species-specific cell adhesion in sponges. Here, we present the structure of the aggregation factor from the marine sponge Microciona prolifera, which constitutes the first description of a circular proteoglycan. We have analyzed chemically dissociated and enzymatically digested aggregation factor with atomic force microscopy, agarose gel electrophoresis, and Western blots using antibodies against the protein and carbohydrate moieties. Twenty units from each of two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms, respectively, stabilized by a hyaluronidase-sensitive component. MAFp3 carries a 200-kDa glycan involved in homologous self-interactions between aggregation factor molecules, whereas MAFp4 carries a 6-kDa glycan that binds cell surface receptors. A 68-kDa lectin found in cell membranes of several sponge species binds the aggregation factor and its protein-free glycans, as well as chondroitin sulfate and hyaluronan. Here, we show that despite their lack of clear sequence homologies with other known proteoglycan structures, the protein and carbohydrate components of sponge aggregation factors assemble to form a supramolecular complex remarkably similar to classical proteoglycans.

Animals↗
PubMed1999

Regulation of plasma membrane blebbing by the cytoskeleton.

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events.

Acrylamide↗
PubMed1999

p150 expression and its prognostic value in squamous-cell carcinoma of the esophagus.

To investigate p150, a recently cloned protein, and its prognostic value in esophageal squamous-cell carcinoma, 100 samples were analyzed immunohistochemically and 30 fresh samples by the immunoblot method. All 100 patients, from Northern China, over-expressed p150 as compared with controls in the immunohistochemical study, and the same was found for the tumors of the 30 patients analyzed by Western-blot analysis. A close correlation could be observed between p150 over-expression and the state of differentiation, since well-differentiated cancers showed high p150 expression, and vice-versa (p < 0.0001). In addition, patients with high p150 expression had significantly fewer tumor metastases (local or distant, p < 0.005). Furthermore, and prognostically more relevant, survival analysis showed that patients with high p150 expression had significantly better overall survival (p < 0.0026). This somehow unexpected result may, however, be typical for squamous carcinoma, since in human cervical carcinoma, another squamous carcinoma, the p150 values were reported to rise early in all tumors, to stay high in patients with longer survival as well. In addition, patients with regional lymph-node metastasis and high p150 expression survived longer than those with regional lymph-node metastasis and lower p150 expression (p < 0.0066). Multivariate analysis showed that p150 is an independent factor for predicting patient survival. The results suggest that p150 may be a new biomarker for squamous-cell carcinoma of the esophagus, with specific prognostic value.

Adult↗
PubMed1999

Cell adhesion and histocompatibility in sponges.

Sponges are the lowest extant metazoan phylum and for about a century they have been used as a model system to study cell adhesion. There are three classes of molecules in the extracellular matrix of vertebrates: collagens, proteoglycans, and adhesive glycoproteins, all of them have been identified in sponges. Species-specific cell recognition in sponges is mediated by supramolecular proteoglycan-like complexes termed aggregation factors, still to be identified in higher animals. Polyvalent glycosaminoglycan interactions are involved in the species-specificity, representing one of the few known examples of a regulatory role for carbohydrates. Aggregation factors mediate cell adhesion via a bifunctional activity that combines a calcium-dependent self-interaction of aggregation factor molecules plus a calcium-independent heterophilic interaction with cell surface receptors. Important cases of cell adhesion are the phenomena involved in histocompatibility reactions. A long-standing prediction has been that the evolutionary ancestors of histocompatibility systems might be found among primitive cell-cell interaction molecules. A surprising characteristic of sponges, considering their low phylogenetic position, is that they possess an exquisitely sophisticated histocompatibility system. Any grafting between two different sponge individuals (allograft) is almost invariably incompatible in the many species investigated, exhibiting a variety of transitive qualitatively and quantitatively different responses, which can only be explained by the existence of a highly polymorphic gene system. Individual variability of protein and glycan components in the aggregation factor of the red beard sponge, Microciona prolifera, matches the elevated sponge alloincompatibility, suggesting an involvement of the cell adhesion system in sponge allogeneic reactions and, therefore, an evolutionary relationship between cell adhesion and histocompatibility systems.

Animals↗
PubMed1999

Regulation of c-met expression in B16 murine melanoma cells by melanocyte stimulating hormone.

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(&agr;) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

Animals↗
PubMed1998

Accumulation in marine sponge grafts of the mRNA encoding the main proteins of the cell adhesion system.

Specific cell adhesion in the marine sponge Microciona prolifera is mediated by an extracellular aggregation factor complex, whose main protein component, termed MAFp3, is highly polymorphic. We have now identified MAFp4, an approximately 400-kDa protein, from the aggregation factor that is translated from the same mRNA as MAFp3. The existence of multiple potential sites for N-glycosylation and calcium binding suggests a direct involvement of MAFp4 in the species-specific aggregation of sponge cells. The deduced partial polypeptide consists of a 16-fold reiterated motif that shows significant similarity to a repeat in an endoglucanase from the symbiontic bacterium Azorhizobium caulinodans and to the intracellular loop of mammalian Na+-Ca2+ exchangers. Restriction fragment length polymorphism analysis indicated that the genomic variability of MAFp4 is high and comparable to that of MAFp3. Their combined polymorphism correlates with allogeneic responses studied in a population of 23 sponge individuals. Peptide mass fingerprinting of tryptic digests of the polymorphic MAFp3 bands observed on polyacrylamide gels after chemical deglycosylation of the Microciona aggregation factor revealed that the variability detected on Southern blots at least partially reflects the individual variability of aggregation factor protein components. Polyclonal antibodies raised against MAFp3 strongly cross-reacted with a 68-kDa protein localized in sponge cell membranes. Immunohistochemical use of the anti-MAFp3 antibodies strongly stained a cell layer along the line of contact in allogeneic grafts. We show that the transcription level of the MAFp3/MAFp4 mRNA in sponge allo- and isografts is clearly increased in comparison with non-grafted tissue. These data are discussed with respect to a possible evolutionary relationship between cell adhesion and histocompatibility systems.

Amino Acid Sequence↗