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Biomedical subjects

G Krohne

Publications and source records attributed to G Krohne.

At least 19 recordsLinked to original sources
PubMed2007

1[alpha],25-dihydroxyvitamin D(3) enhances annexin II dependent proliferation of osteoblasts.

Cells experience a variety of physiological and non-physiological stresses and consequently have appropriate mechanisms to deal with such deviations from homeostasis. Particularly subject to mechanical stress and shear forces are the cells that make up the bones. Osteoblastic cells can interpret this stress as a stimulus for proliferation; however, the molecular mechanisms underlying this phenomenon are poorly understood. We have identified annexin II as being specifically upregulated in mechanically stressed osteoblasts and found that increased levels of this protein are necessary for 1[alpha],25-dihydroxyvitamin D(3) mediated augmentation of the proliferative response of osteoblasts after mechanical stress. Our data demonstrate a novel interaction between 1[alpha],25-dihydroxyvitamin D(3) and annexin II in the proliferative response of osteoblasts as well as a novel function for annexin II in the stress response. These findings may offer new therapeutic opportunities for conditions that require regenerative osteoblastic activity such as osteoporosis.

Adult↗
PubMed2003

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis.

The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.

Chlorophyta↗
PubMed2000

The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance.

Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.

Animals↗
PubMed2000

Xylem conduits of a resurrection plant contain a unique lipid lining and refill following a distinct pattern after desiccation.

The axial and radial refilling with water of cut dry branches (up to 80 cm tall) of the resurrection plant Myrothamnus flabellifolia was studied in both acro- and basipetal directions by using 1H-NMR imaging. NMR measurements showed that the conducting elements were not filled simultaneously. Axial water ascent occurred initially only in a cluster of a very few conducting elements. Refilling of the other conducting elements and of the living cells was mainly achieved by radial extraction of water from these initial conducting elements. With time, xylem elements in a few further regions were apparently refilled axially. Radial water spread through the tissue occurred almost linearly with time, but much faster in the acropetal than in the basipetal direction. Application of hydrostatic pressure (up to 16 kPa) produced similar temporal and spatial radial refilling patterns, except that more conducting elements were refilled axially during the first phase of water rise. The addition of raffinose to the water considerably reduced axial and radial spreading rates. The polarity of water climbing was supported by measurements of the water rise in dry branches using the 'light refraction'(and, sometimes, the 'leaf recurving') method. Basipetal refilling of the xylem conduit exhibited biphasic kinetics; the final rise height did not exceed 20-30 cm. Three-cm-long branch pieces also showed a directionality of water climbing, ruling out the possibility that changes in the conducting area from the base to the apex of the branches were responsible for this effect. The polarity of water ascent was independent of gravity and also did not change when the ambient temperature was raised to c. 40 degrees C. At external pressures of 50-100 kPa the polarity disappeared, with basipetal and acropetal refill times of the xylem conduit of tall branches becoming comparable. Refilling of branches dried horizontally (with a clinostat) or inverted (in the direction of gravity) showed a pronounced reduction of the acropetal water rise to or below basipetal water climbing level (which was unaffected by this treatment). Unlike water, benzene and acetone climbing showed no polarity. In the case of benzene, the rise kinetics (including the final heights) were comparable with those measured acropetally for water, whereas with acetone the rise height was less. Transmission electron microscopy of dry branches demonstrated that the inner surfaces of the conducting tracheids and vessels were lined with a continuous osmiophilic (lipid) layer, as postulated by the kinetic analysis and light microscopy studies. The thickness of the layer varied between 20 and 80 nm. The parenchymal and intervessel pits as well as numerous tracheid corners contained opaque inclusions, presumably also consisting of lipids. Electron microscopy of rehydrated plants showed that the lipid layer was either thinned or had disintegrated and that numerous vesicle-like structures and lipid bodies were formed (together with various intermediate structural elements). These, many other data and the physical-chemical literature imply that several (radial) driving forces (such as capillary condensation, Marangoni forces, capillary, osmotic and turgor pressure forces) operate when a few conducting elements become axially refilled with water. These forces apparently lead to an avalanche-like radial refilling of most of the conducting elements and living cells, and thus to the removal of the 'internal cuticle' and of the hydrophobic inclusions in the pits. The polarity of water movement presumably results from high resistances in the basipetal direction, which are created by local gradients in the thickness of the lipid film as a result of draining under gravity in response to drought. There are striking similarities in morphology and function between the xylem-lining lipid film and the lung surfactant film lining the pulmonary air spaces of mammals.

Acetone↗
PubMed2000

Localization of glucocorticoid hormone receptors in mitochondria of human cells.

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.

Animals↗
PubMed1999

Distribution of emerin during the cell cycle.

Human emerin is a nuclear membrane protein that is lost or altered in patients with Emery-Dreifuss muscular dystrophy (EMD). While the protein is expressed in the majority of human tissues analyzed, the pathology predominates in cardiac and skeletal muscles of patients with EMD. Our results show that emerin can be detected by immunocytochemistry and immunoblotting in the nuclear envelope of all vertebrates studied from man to Xenopus. Immunolocalizations and nuclear envelope extraction experiments confirm that emerin possesses properties characteristic for integral membrane proteins of the inner nuclear membrane. Some nuclear envelope proteins are localized also in annulate lamellae (AL), i.e. cytoplasmic flattened membrane cisternae penetrated by pore complexes. To verify whether emerin is contained in these membrane stacks, we have induced the formation of AL by exposure of rat cells (line RV-SMC) to sublethal doses of the antimitotic drug vinblastine sulfate and found that emerin is present in the nuclear envelope, but is absent from AL. In contrast to the homogeneous distribution of emerin in the nuclear envelope of interphase cells, this protein shows a focal accumulation in the nuclear membranes of late telophase cells. During early reassembly of the nuclear envelope at this mitotic stage emerin colocalizes with lamin A/C but not with lamin B and LAP2 proteins. Confocal laser scanning microscopy after double-labeling experiments with emerin and tubulin shows that emerin is concentrated in areas of the mitotic spindle and in the midbody of mitotic cells suggesting a close interaction of these proteins. Our data suggest that emerin participates in the reorganisation of the nuclear envelope at the end of mitosis.

Animals↗
PubMed1999

Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs.

The p58/lamin B receptor of vertebrates is localized in the inner nuclear membrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast majority of this membrane protein is localized in cytoplasmic membranes. Only very small amounts of p58 not detectable by immunofluorescence microscopy were contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nuclei reconstituted in vitro in Xenopus egg extracts contained p58, and the nucleoplasmic domain of Xp58 could be specifically bound to sperm chromatin in vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane whereas the complement of lamins is identical in both cell types. To gain insight into the properties of oocyte p58 we microinjected isolated nuclei of cultured rat cells into the cytoplasm of Xenopus oocytes. The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nuclear membrane of rat nuclei. Our data indicate that the peripheral chromatin but not lamins are required for the retention of p58 in the inner nuclear membrane. Sucrose step gradient centrifugation of total oocyte membranes revealed that the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertilized eggs were found in the same fractions. By electron microscopical immunolocalizations one major population of meiotic p58 vesicles was identified that contained exclusively p58 and a second minor population (ca. 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins. Egg vesicles containing pore membrane proteins were predominantly recovered in gradient fractions that did not contain p58 and B-type lamins. Our data indicate that the targeting of p58 to chromatin at the end of mitosis in the early Xenopus embryo is a process independent from that of lamin targeting. Comparable to the situation in oocytes and eggs, a significant proportion of p58 of interphase cells could be recovered in fractions that did not contain lamins. This population of p58 molecules could be extracted from A6-cells with buffers containing 1% Triton X-100/0.15 M NaCl and could be pelleted by a 50,000 g centrifugation. A- and B-type lamins were not detectable in the p58 containing pellet.

Amino Acid Sequence↗
PubMed1999

Molecular characterization and developmentally regulated expression of Xenopus lamina-associated polypeptide 2 (XLAP2).

Lamina-associated polypeptides 2 (LAP2alpha, beta, gamma)/thymopoietins (TPalpha, beta, gamma) are a family of proteins that are generated by alternative splicing from a single gene. These proteins have been primarily characterized in mammals. One member of this protein family, the integral membrane protein LAP2beta/TPbeta, has been localized to the inner nuclear membrane of somatic cells where it binds to chromatin and B-type lamins. By cDNA cloning we have characterized XLAP2, a Xenopus homologue of the mammalian LAP2beta. Using LAP2-specific antibodies, the Mr 68,000 XLAP2 was found to be the only member of the LAP2/TP family expressed in somatic cells and adult tissues. XLAP2 was not detected in oocytes, eggs and in early embryos up to the gastrula stage at the mRNA and protein level demonstrating that it is not synthesized from maternal mRNA. In counterpart oocytes, eggs, and embryos contained one LAP2-related integral membrane proteins of Mr 84,000. Northern blot analysis with the XLAP2 cDNA showed that a single hybridizing mRNA band of 1.8-2.0 kb was present in Xenopus somatic cells whereas two other hybridizing mRNA species of 2.8-3.0 and 0. 9-1.1 kb were present in oocytes, eggs and early embryos. All together, these results indicated that at least three distinct LAP2-related proteins might be expressed in Xenopus. The LAP2/TP protein of Mr 84,000 is present in the early embryos but its amount decreases during embryogenesis concomitant with the increase of XLAP2 in the embryo. Our results are the first description of the developmentally regulated expression of integral nuclear envelope proteins during early embryogenesis.

Amino Acid Sequence↗
PubMed1998

Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP.

We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms.

Adenosine Triphosphate↗
PubMed1998

Human mast cells augment fibroblast proliferation by heterotypic cell-cell adhesion and action of IL-4.

Mast cells have been implicated in the pathogenesis of fibrosis because of their increased number in chronic inflammatory reactions. In a previous study, we had shown that human mast cells readily attach and form heterotypic cell-cell contacts when seeded on top of fibroblast monolayers. Here, we report that human mast cells stimulate fibroblast proliferation after cell-cell contact. Proliferation was measured by 5-bromo-2'-deoxyuridine or [3H]thymidine uptake of subconfluent fibroblast monolayers after attachment of mast cells that had been preincubated with mitomycin C. An 18-h coculture of the human mast cell line HMC-1 doubled proliferation of normal skin fibroblasts. Moreover, normal mast cells prepared from neonatal foreskin doubled fibroblast proliferation. The stimulatory effect was dependent on heterotypic cell-cell contact since it was not transferred by tissue culture supernatants from mast cells. We hypothesized that mast cell cytokines secreted after heterotypic cell-cell contact stimulate fibroblast proliferation. Several mast cell-derived cytokines were tested for effects on fibroblast proliferation. Only IL-4 was able to double fibroblast proliferation. Additional experiments revealed that: 1) the stimulatory effect of IL-4 as well as of the mast cell coculture could be completely abrogated by preincubation of fibroblasts with an anti-IL-4R mAb blocking ligand binding; 2) mast cell-derived IL-4 acts as a second signal for fibroblasts since it amplifies the action of low doses of obligatory fibroblast growth factors such as fibroblast growth factor or platelet-derived growth factor.

Cell Communication↗
PubMed1998

Assembly of Drosophila lamin Dm0 and C mutant proteins studied with the baculovirus system.

Despite extensive knowledge of the in vitro polymerization properties of nuclear lamins, it is still not well understood how the nuclear lamina assembles in vivo. To learn more about the relationship between in vitro and in vivo polymerization of nuclear lamins, we expressed Drosophila lamin Dm0, mutant proteins, having well defined alterations of their in vitro polymerization properties, in Sf9 cells using the baculovirus system. All lamin Dm0 mutants assembled into fibrillar aggregates indistinguishable in morphology from those assembled by the wild-type protein. However, in contrast to wild-type lamin Dm0, mutant proteins were extracted with buffers of physiological ionic strength and pH containing Triton X-100. These results indicate that various types of lamin dimer-dimer interactions can be disrupted without affecting the morphology of the lamin Dm0 polymer. However, all types of dimer-dimer interactions tested appear to be important for full polymer stability. In addition, we analyzed the polymer formation of two Drosophila lamin C mutants and found that a segment in the carboxy-terminal tail domain is required for assembly of lamin C paracrystals at the nuclear lamina.

Animals↗
PubMed1997

Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44.

The three-step model of cell migration consisting of protrusion of a leading lamella, attachment to the substrate, and contraction of the cell body is well established for fibroblasts migrating across planar surfaces. However, it is not resolved to what extent the migration of cancer cells in a 3-dimensional tissue environment follows similar principles. Here, we present evidence that the migration of highly invasive MV3 melanoma cells in 3-dimensional collagen matrices follows the three-step concept of migration but also results in characteristic reorganization of the extracellular matrix. After incorporation in the lattice, MV3 cells spontaneously developed a slow type of migration (mean velocity, 0.19 microm/min), leading to alignment of collagen fibers at attachment sites, as detected from unfixed and fixed samples by confocal reflection contrast in combination with immunofluorescence staining. In the process of migration, the formation of focal clusters or stripes of alpha2 and beta1 integrins colocalized with binding sites to collagen fibrils at the leading as well as the trailing edge. In contrast, CD44 was nonclustered and redistributed toward the rear end of the cell. At detachment sites, dynamic fiber traction, localized fiber disruption, and the release of cell surface determinants, including alpha2beta1 integrins and CD44, resulted in circumscribed matrix reorganization. Not infrequently, these emerging tube-like paths of least resistance bordered by a dense fiber network facilitated the reorientation and contact guidance of proximate MV3 cells to migrate along the preexisting path. In conclusion, the migration of MV3 cells in 3-dimensional collagen lattices resulted in dynamic tissue reorganization and receptor shedding the consequences of which were directly visualized by combining confocal reflection imaging with immunofluorescence.

Animals↗
PubMed1997

Assembly of A- and B-type lamins studied in vivo with the baculovirus system.

We have expressed an A-type lamin (Xenopus lamin A), a probable A-type lamin (Drosophila lamin C), two B-type lamins (Xenopus lamin LI, Drosophila lamin Dmo), and two mutants of Xenopus lamin A in Sf9 cells. All proteins were synthesized at high levels resulting in formation of paracrystals with an axial repeat of 18.5-20.0 nm by A-type lamins; in contrast B-type lamins assembled into aggregates with a fibrillar ultrastructure. Of the four wild-type proteins analyzed only lamin Dmo was found in the nuclear compartment of Sf9 cells in association with the lamina whereas the three other lamins assembled into polymers localized in the cytoplasm as well as the nucleoplasm. The Xenopus lamin A mutant lacking the complete carboxy-terminal tail assembled in the cytoplasm into long filament bundles consisting of fibrils of less than 6 nm diameter. In vitro the non-helical amino-terminal head domain of lamins is required for the formation of 'head-to-tail' polymers. A lamin A mutant lacking this domain could be efficiently extracted from Sf9 cells with physiological buffers containing Triton X-100, demonstrating the importance of this domain for lamin assembly in vivo.

Animals↗
PubMed1996

Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization.

Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod-shaped Gram-negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus- and species-specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the gamma-subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the gamma-subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co-evolution of the symbiotic partners and a vertical transmission of the bacteria.

Animals↗
PubMed1996

Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation.

We have previously shown that Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII, and that portions of each are associated with distinct egg vesicle populations. We now report that a lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. We have quantitated the three B-type lamins per oocyte nucleus, and have calculated relative ratios of LI:LIII = 1:100, and LII:LIII = 1:10. Similar to lamin LII, 5-15% of lamin LI is associated with egg membranes in a biochemically stable manner. Egg vesicles absorbed with lamin isoform-specific antibodies to magnetic beads indicate that lamin LI-associated egg membranes are of heterogenous morphology, and are independent from the lamin LII and LIII vesicle populations. Compared to other nuclear envelope proteins, the synthesis of lamin LI protein is specifically elevated during meiotic maturation, resulting in a 4- to 12-fold higher amount of lamin LI in eggs than is present in oocyte nuclei. Immunofluorescence and immunoblot analysis demonstrated that lamins LI, LII, and LIII are associated with the nuclear envelope formed on demembranated sperm when added to activated egg extract. These results strongly suggest that three different lamin-associated vesicle populations are involved in the formation of a nuclear envelope in egg extracts.

Animals↗