Search PubMed⌕ Search

Biomedical subjects

Georg Krohne

Publications and source records attributed to Georg Krohne.

At least 19 recordsLinked to original sources
PubMed2007

Molecular characterization of Vibrio harveyi bacteriophages isolated from aquaculture environments along the coast of India.

Seven bacteriophages specific to Vibrio harveyi, the causative agent of luminous vibriosis in shrimp, were isolated from coastal aquaculture systems like shrimp farms, hatcheries and tidal creeks along the east and west coast of India. All the seven phages were found to have the typical head and tail morphology with double-stranded DNA as genetic material. Morphologically, six phages were grouped under family Siphoviridae and one under Myoviridae. These phages were further characterized with respect to host range, morphology and structural proteins. Genomic fingerprinting was carried out using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD). Major capsid proteins of all the phages detected by SDS-PAGE were distinct from one another. All the phages were found to be highly lytic for V. harveyi and had different lytic spectrum for the large number of isolates tested. Six of the seven phages isolated had a broad lytic spectrum and could be potential candidates for biocontrol of V. harveyi in aquaculture systems.

Aquaculture↗
PubMed2006

Structural and functional characterization of the zebrafish lamin B receptor.

The lamin B receptor (LBR) is an integral membrane protein of the inner nuclear membrane that is interacting with B-type lamins, chromatin and DNA. The complete loss of the protein in mouse mutants causes a reduced viability of embryos, and viable animals develop abnormalities of the skeleton. Here, we present the molecular characterization of the zebrafish LBR (zLBR) gene and the functional analysis of LBR during zebrafish embryogenesis. We found that the coding region of the LBR mRNA of zebrafish as well as of mammals is contained in 13 exons. At the protein level, human and zebrafish LBR exhibit a high sequence identity (57% and higher) in 8 of the 13 exons. Knockdown of zLBR by microinjection of 0.5-1.0 mM morpholino antisense oligonucleotides (MO) into 1- to 2-cell stage embryos reduced the amount of endogenous zLBR protein to approximately 10-20%. The viability of MO-injected embryos within 24 h was reduced to 70-77%. Surviving 1-day-old embryos exhibited morphological alterations including reduced growth of head structures, retardation of tail growth and a bent backbone and tail. Expression analysis of the transcription factors no tail (ntl) and goosecoid (gsc) by in situ hybridization suggests that these malformations are caused by altered cell migration during gastrulation. Our data indicate that the LBR of zebrafish and mammals are both required for correct development.

Amino Acid Sequence↗
PubMed2006

Developmental control of nuclear size and shape by Kugelkern and Kurzkern.

BACKGROUND: The shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization. RESULTS: We identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane. CONCLUSIONS: Kugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.

Amino Acid Motifs↗
PubMed2006

Measles virus contact with T cells impedes cytoskeletal remodeling associated with spreading, polarization, and CD3 clustering.

CD3/CD28-induced activation of the PI3/Akt kinase pathway and proliferation is impaired in T cells after contact with the measles virus (MV) glycoprotein (gp) complex. We now show that this signal also impairs actin cytoskeletal remodeling in T cells, which loose their ability to adhere and to promote microvilli formation. MV exposure results in an almost complete collapse of membrane protrusions associated with reduced phosphorylation levels of cofilin and ezrin/radixin/moesin (ERM) proteins. Consistent with their inability to activate Cdc42 and Rac1 in response to the ligation of CD3/CD28, T cells exposed to MV fail to acquire a morphology consistent with spreading and lamellopodia formation. In spite of these impairments of cytoskeleton-driven morphological alterations, these cells are recruited into conjugates with dendritic cells as efficiently as control T cells. The signal elicited by MV, however, prevents T cells to polarize as documented by a failure to redistribute the microtubule organizing center toward the synapse. Moreover, CD3 cannot be efficiently clustered and redistributed to the central region of the immunological synapse. Thus, by inducing microvillar collapse and interfering with cytoskeletal remodeling, MV signaling disturbs the ability of T cells to adhere, spread, and cluster receptors essential for sustained T-cell activation.

Actins↗
PubMed2005

The Drosophila melanogaster LEM-domain protein MAN1.

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.

Amino Acid Motifs↗
PubMed2005

Maurer's clefts: a novel multi-functional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes.

Discovered in 1902 by Georg Maurer as a peculiar dotted staining pattern observable by light microscopy in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum, the function of Maurer's clefts have remained obscure for more than a century. The growing interest in protein sorting and trafficking processes in malarial parasites has recently aroused the Maurer's clefts from their deep slumber. Mounting evidence suggests that Maurer's clefts are a secretory organelle, which the parasite establishes within its host erythrocyte, but outside its own confines, to route parasite proteins across the host cell cytoplasm to the erythrocyte surface where they play a role in nutrient uptake and immune evasion processes. Moreover, Maurer's clefts seem to play a role in cell signaling, merozoite egress, phospholipid biosynthesis and, possibly, other biochemical pathways. Here, we review our current knowledge of the ultrastructure of Maurer's clefts, their proteinaceous composition and their function in protein trafficking.

Cytoplasm↗
PubMed2005

Microbial diversity of the freshwater sponge Spongilla lacustris.

To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alpha proteobacteria (n=19), the Actinobacteria (n=15), the Beta proteobacteria (n=2), and the Chloroflexi (n=2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and beta proteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alpha proteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.

Animals↗
PubMed2005

The myristoylation site of meiotic lamin C2 promotes local nuclear membrane growth and the formation of intranuclear membranes in somatic cultured cells.

Lamin C2 is a splice product of the mammalian lamin A gene and expressed in primary spermatocytes where it is distributed in the form of discontinuous plaques at the nuclear envelope. We have previously shown that the aminoterminal hexapetide GNAEGR of lamin C2 following the start methionine is essential for its association with the nuclear envelope and that the aminoterminal glycine of the hexapeptide is myristoylated. Here we have analyzed the ultrastructural changes induced in COS-7 and Xenopus A6 cells by overexpressing rat lamin C2 or a human lamin C mutant possessing the lamin C2-specific hexapeptide at its aminoterminus. Both lamins were targeted to the nuclear envelope of mammalian and amphibian cells and induced the formation of intranuclear membranes, whereas wild-type human lamin C and a lamin C2 mutant, that both lack this lipid moiety, did not. Our data indicate that the myristoyl group of lamin C2 has besides its demonstrated role in nuclear envelope association additional functions during spermatogenesis. Our present study complements previously published results where we have shown that the CxxM motif of lamins promotes nuclear membrane growth (Prüfert et al., 2004. J. Cell Sci. 117, 6105-6116).

Animals↗
PubMed2004

The lamin CxxM motif promotes nuclear membrane growth.

We analyzed the influence of lamins on nuclear envelope growth in cultured Xenopus A6 cells by the overexpression of human lamin A, Xenopus and zebrafish lamins B2 and Drosophila lamins Dm0 and C as GFP fusion proteins. Lamins containing a CxxM motif in their primary sequence (lamins A, B2, Dm0) induced the formation of lobulated nuclei with multi-membrane-layered, highly folded nuclear membranes and intranuclear membrane assemblies, as observed by electron microscopy. Such morphological alterations were not observed with Drosophila lamin C, a lamin without this motif or with a lamin B2 mutant (B2-SxxM) where the cysteine of the CxxM motif is replaced by a serine. Drosophila lamin C mutants containing a CxxM motif behaved like B-type lamins thus confirming that this tetrapeptide is directly involved in the morphological changes we observed. Nuclear membrane proliferation could also be induced by lamin B2 in COS-7 cells and in zebrafish embryos but not by human lamin A in COS-7 cells. We speculate that the human lamin A is incompletely processed in Xenopus A6 cells and therefore behaves in this cell line like a B-type lamin. Our results indicate that the CxxM motif of B-type lamins has a dual function: it mediates lamin targeting to the inner nuclear membrane thereby promoting nuclear membrane growth.

Amino Acid Motifs↗
PubMed2004

The lamin B receptor of Drosophila melanogaster.

The lamin B receptor (LBR) is an integral membrane protein of the inner nuclear membrane that has so far been characterized only in vertebrates. Here, we describe the Drosophila melanogaster protein encoded by the annotated gene CG17952 that is the putative ortholog to the vertebrate LBR. The Drosophila lamin B receptor (dLBR) has the following properties in common with the vertebrate LBR. First, structure predictions indicate that the 741 amino acid dLBR protein possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments in the C-terminal domain of the molecule. Second, immunolocalization and cell fractionation reveal that the dLBR is an integral membrane protein of the inner nuclear membrane. Third, dLBR can be shown by co-immunoprecipitations and in vitro binding assays to bind to the Drosophila B-type lamin Dm0. Fourth, the N-terminal domain of dLBR is sufficient for in vitro binding to sperm chromatin and lamin Dm0. In contrast to the human LBR, dLBR does not possess sterol C14 reductase activity when it is expressed in the Saccharomyces cerevisiae erg24 mutant, which lacks sterol C14 reductase activity. Our data raise the possibility that, during evolution, the enzymatic activity of this insect protein had been lost. To determine whether the dLBR is an essential protein, we depleted it by RNA interference in Drosophila embryos and in cultured S2 and Kc167 cells. There is no obvious effect on the nuclear architecture or viability of treated cells and embryos, whereas the depletion of Drosophila lamin Dm0 in cultured cells and embryos caused morphological alterations of nuclei, nuclear fragility and the arrest of embryonic development. We conclude that dLBR is not a limiting component of the nuclear architecture in Drosophila cells during the first 2 days of development.

Amino Acid Sequence↗
PubMed2004

Lamins.

Explore the source record for details and available documents.

Animals↗
PubMed2004

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel).

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.

Alternative Splicing↗
PubMed2004

The lamina-associated polypeptide 2 (LAP2) genes of zebrafish and chicken: no LAP2alpha isoform is synthesised by non-mammalian vertebrates.

The mammalian lamina-associated polypeptide 2 (LAP2) gene encodes six isoforms (LAP2alpha, beta, delta, epsilon, gamma, zeta) that are synthesised from alternatively spliced mRNAs. The mammalian LAP2alpha is one of the predominant isoforms and localised in the nucleoplasm whereas LAP2beta, delta, epsilon, and gamma are integral membrane proteins of the inner nuclear membrane. We have analysed the LAP2 gene structure of the zebrafish Danio rerio as an attractive lower vertebrate model organism. The zebrafish LAP2 (ZLAP2) gene without regulatory sequences spans approximately 19 kb of genomic DNA. It contains 15 exons that encode the isoforms ZLAP2beta, gamma, and omega which are localised in the inner nuclear membrane. By radiation hybrid mapping, we have located the gene onto linkage group 4 between EST markers fc01g04 (213.97cR) and fb49f01 (215.69cR). The identification of a chicken genomic clone comprising the complete coding region of the avian LAP2 gene enabled us to compare the LAP2 gene structure amongst vertebrates. In contrast to the mammalian LAP2 gene, the zebrafish and the chicken sequences do not encode for an alpha-isoform. In parallel we searched for an alpha-isoform in birds using polyclonal and monoclonal LAP2 antibodies specific for the common evolutionary conserved aminoterminal domain present in all isoforms. We detected LAP2beta as the predominant isoform but no LAP2alpha in tissues of 10-day-old chicken embryos and cultured chicken fibroblasts thus confirming the genomic analysis. The comparison of each zebrafish and chicken LAP2 exon with the corresponding exons of the human LAP2 gene demonstrates that the degree of identity at the amino acid level is much higher between the human and chicken than between the human and zebrafish sequences. By Blast search with the nucleotide and amino acid sequences of the human LAP2alpha, we did not find any significant homologies in databases of the zebrafish and chicken sequences. Our data suggest that LAP2alpha is a novelty of mammals.

Amino Acid Sequence↗
PubMed2004

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections.

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.

Animals↗
PubMed2003

The lamina-associated polypeptide 2 (LAP2) isoforms beta, gamma and omega of zebrafish: developmental expression and behavior during the cell cycle.

Zebrafish lamina-associated polypeptides 2 (ZLAP2) beta, gamma and omega have in common an N-terminal region with a LEM domain, and in the C-terminal half of the molecule a lamina binding domain and a membrane spanning sequence. The maternally synthesized omega is the largest isoform and the only LAP2 present in the rapidly dividing embryonic cells up to the gastrula stage. ZLAP2omega levels decrease during development, concomitant with the increase of the somatic isoforms ZLAP2beta and gamma. In somatic zebrafish cells ZLAP2gamma is the predominant isoform, whereas only small amounts of ZLAP2beta are present. During early embryonic development, ZLAP2omega becomes associated with mitotic chromosomes before anaphase. The surface of these chromosomes is decorated with vesicles, and each chromosome assembles its own nuclear envelope at the end of mitosis (karyomere formation). Ectopically expressed ZLAP2omega-green fluorescent protein (GFP) fusion protein targets vesicles to mitotic chromosomes in Xenopus A6 cells, suggesting that ZLAP2omega is involved in karyomere formation during early zebrafish development. When ZLAP2beta and gamma were expressed as GFP fusion proteins in Xenopus A6 cells, the beta- but not the gamma-isoform was found in association with mitotic chromosomes, and ZLAP2beta-containing chromosomes were decorated with vesicles. Further analysis of ZLAP2-GFP fusion proteins containing only distinct domains of the ZLAP2 isoforms revealed that the common N-terminal region in conjunction with beta- or omega-specific sequences mediate binding to mitotic chromosomes in vivo.

Amino Acid Sequence↗