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Biomedical subjects

B Humbel

Publications and source records attributed to B Humbel.

11 recordsLinked to original sources
PubMed1995

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so-called SH2 domains. The signal transduction pathways activated by EGF include the phosphatidylinositol pathway, leading to activation of protein kinase C and to increase in the intracellular Ca2+ concentration, and to the ras pathway leading to MAP kinase activation. Recently the cytoplasm has been implicated as playing an important role in EGF induced signal transduction. The EGF receptor has been demonstrated to be an actin-binding protein. In addition EGF causes a rapid actin depolymerisation and the formation of membrane ruffles. In particular these membrane ruffles have been shown to act as the first site of signal transduction after EGF binding, and thus may be considered as signal transduction structures. Finally evidence has been presented suggesting a positive role for EGF and/or the receptor in the nucleus.

Amino Acid Sequence↗
PubMed1994

Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells.

When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the heterodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.

Actins↗
PubMed1993

Properties of bacteriorhodopsin derivatives constructed by insertion of an exogenous epitope into extra-membrane loops.

Bacteriorhodopsin (BR) is folded into a bundle of seven alpha-helices which is embedded in the cellular membrane of Halobacterium salinarium; these helices are connected by short extra-membrane loops, three on the cytoplasmic side and three on the outside. Oligonucleotide-directed insertion or replacement mutagenesis was used to integrate the C-terminal sequence (13 amino acids long) of Sendai virus L-protein individually into each of the six helix-connecting loops. The altered gene products were obtained by expression of the mutant genes in either Escherichia coli or Schizosaccharomyces pombe and were used to reconstitute BR in proteoliposomes. In four cases (altered loops B/C, C/D, D/E or E/F), the mutant BRs were found to be fully functional as judged by light-driven proton pumping and photocycle kinetics. Within the four functional BR variants, recognition of the viral epitope by a monoclonal antibody is restricted to modified loops B/C and E/F. Immunogold staining of S.pombe cells producing either of the two latter BR variants shows that the protein is distributed among various cellular membranes but is not present in mitochondrial membranes. Sequence alteration of loop A/B or F/G resulted in loss of function, most plausibly due to a folding defect of the respective proteins. These results on the one hand document differences in structural importance of the various BR extra-membrane loops and on the other hand open the door to the construction of multifunctional membrane proteins via loop replacement mutagenesis of BR.

Amino Acid Sequence↗
PubMed1992

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Amino Acid Sequence↗
PubMed1992

Immunoelectron microscopic localization of microsomal alanine aminopeptidase.

The localization of microsomal alanine aminopeptidase was investigated in the rat kidney. Resin embedding failed to demonstrate the localization of the enzyme by immunogold labelling. Using a cryo-ultramicrotomy method the enzyme could be detected on the luminal side of the brush border membrane of proximal tubular cells and to a lesser degree in their mitochondria. Furthermore, vesicular structures labelled with gold were found in the cytoplasma in the apical region of these cells.

Aminopeptidases↗
PubMed1992

A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies.

We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus. By immunoblotting mAb 5E10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5. Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium. In the human bladder carcinoma cell line T24 the number of nuclear spots were found to be 21 +/- 10 (n = 132). In many cells spots were found also in the cytoplasm. In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots. Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes. The spherical structures that contain the antigen were tightly bound to the nuclear matrix. Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 microns diameter spherical, electron-dense structures, reminiscent of nuclear bodies. Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres. The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies. On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies.

Animals↗
PubMed1991

The nucleus: a black box being opened.

Until recently our knowledge about the structural and functional organization of the cell nucleus was very limited. Recent technical developments in the field of ultrastructural analysis, combined with ongoing research on the properties of the nuclear matrix, give new insight into how the nucleus is structured. Two types of observations shape our ideas about nuclear organization. First, most nuclear functions (replication, transcription, RNA processing, and RNA transport) are highly localized within the nucleus, rather than diffusely distributed. Moreover, they are associated with the nuclear matrix. Second, chromatin is organized in discrete loops, bordered by nuclear matrix attachment sequences (MARs). Each loop may contain one or several genes. The arrangement of chromatin in loops has profound consequences for the regulation of gene expression.

Animals↗
PubMed1987

Calcium-binding proteins in carcinoma, neuroblastoma and glioma cell lines.

Antisera against the Ca2+-binding proteins parvalbumin, calbindin D-28K, and the S-100 proteins were used to study the distribution of their target proteins in selected human carcinoma (LICR-HN6;Caco-2), mouse neuroblastoma (clone NB-2a), and rat glioma cell lines (clone C-6). Pronounced staining with anti-parvalbumin was observed in the cytosol of all cells as well as in some nuclei, in particular, mitotic nuclei were highly immuno-reactive. Applying light and immune-electron microscopy (colloidal gold labelling) the parvalbumin-fluorescence was associated with filaments in the LICR-HN6 cells. However, this immunoreactivity was not a result of the presence of parvalbumin itself--as shown by biochemical analyses (HPLC, 2D-PAGE)--but was due to the presence of a Ca2+-binding and tumour-associated protein with similar biochemical and immunological properties. S-100 proteins were present in all tumour cell lines but their intracellular distribution was different from calbindin D-28K. Calbindin-immunoreactivity was found on the membranes of the carcinoma cell lines whereas neuroblastoma and glioma cells remained unlabelled. It is suggested that these proteins might be involved in the modulation of the enhanced stimulation of Ca2+-dependent processes occurring in tumour cells.

Animals↗
PubMed1986

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils.

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.

Animals↗