Journal of Cancer Research and Clinical Oncology, Oct 1, 2001
Keyhole limpet hemocyanin (KLH) is a wellestablished immune stimulant and hapten carrier, and Hal... more Keyhole limpet hemocyanin (KLH) is a wellestablished immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1, KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture,. amino acid sequence, gene structure, and recombinant production of KLH and HtH. Abbreviations FU functional unit • HtH Haliotis tuberculata hemocyanin • KLH keyhole limpet hemocyanin • OdH Octopus dofleini hemocyanin R4
To cite: Lieb B, Schmitt P. Randomised double-blind placebo-controlled intervention study on the ... more To cite: Lieb B, Schmitt P. Randomised double-blind placebo-controlled intervention study on the nutritional efficacy of a food for special medical purposes (FSMP) and a dietary supplement in reducing the symptoms of veisalgia.
Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canon... more Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canonical 110 kDa form of somatic cells (Richard and Bogenhagen, Dev. Biol. 146: 4-11, 1991). By immunofluorescence microscopy using variant-specific antibodies we show that this high molecular weight form is associated with lampbrush chromosome loops and the inner regions of the amplified nucleoli. Inhibition of topo I-activity by either Camptothecin-treatment or microinjection of neutralizing antibodies resulted in loop retraction and the condensation of chromosomes and amplified nucleoli. These data indicate that the oocyte-specific 165 kDa form of topo I is involved in transcriptional processes mediated by RNA polymerase I and II and is therefore functionally equivalent to the somatic cell 110 kDa counterpart.
Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastro... more Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the ''slit apparatus'', local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20620 nm. In blueblooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposedthough not proven-that in the rare red-blooded snail species they might synthesize and secrete the hemoglobin. However, the cellular secretion pathway for respiratory proteins, and the functional role(s) of the enigmatic rhogocyte slit apparatus are still unclear. Additional functions for rhogocytes have been proposed, notably a role in protein uptake and degradation, and in heavy metal detoxification. Here we provide new structural and functional information on the rhogocytes of the redblooded freshwater snail Biomphalaria glabrata. By in situ hybridization of mantle tissues, we prove that rhogocytes indeed synthesize hemoglobin. By electron tomography, the first three dimensional (3D) reconstructions of the slit apparatus are provided, showing detail of highly dense material in the cytoplasmic bars close to the slits. By immunogold labelling, we collected evidence that a major component of this material is actin. By genome databank mining, the complete sequence of a B. glabrata nephrin was obtained, and localized to the rhogocytes by immunofluorescence microscopy. The presence of both proteins fit the ultrastructure-based hypothesis that rhogocytes are related to mammalian podocytes and insect nephrocytes. Reactions of the rhogocytes to deprivation of food and cadmium toxification are also documented, and a possible secretion pathway of newly synthesized respiratory proteins through the slit apparatus is discussed.
We have studied individual keratins of an elasmobrancb, the shark Scyliorhinus stellaris (the les... more We have studied individual keratins of an elasmobrancb, the shark Scyliorhinus stellaris (the lesser-spotted dogfish). From various shark tissues, notably skin and stomach, cytoskeletal proteins were isolated and then separated by two-dimensional polyacrylamide gel electrophoresis. Using complementary keratin blot-binding assays and immunoblotting, among these proteins we identified a variety of type I and type II keratins. According to their tissue-specific expression, we distinguished Is and lIs keratins from IE and lIE keratins ("S" and "E" from "simple epitbelial" and "epidermal", respectively). Guinea pig antibodies which in immunoblots specifically labeled the entire range of identified shark keratins, and a monoclonal antibody specific for IE keratins were used for immunoOuorescence microscopy of a broad range of shark tissues. These experiments demonstrated that in this shark, keratin expression is largely restricted to epithelia and-in contrast to the situation in teleost fishes-is lacking in mesenchymally derived cells and tissues. Peptide mass mapping of the major electrophoretically separated shark keratin spots revealed that the identified Is, lIs and lIE polypeptides are modifications of a single genuine keratin, respectively, wbereas there are two different IE keratins. It, therefore, appears that in this shark most (if not all) of the keratin cytoskeleton is constituted by only five different gene products (each present in various modifications): a heterologous pair of "S" and three different "E" keratins. We sequenced three of them (Is, lIs and lIE) via eDNA Abbreviations. CKBB Complementary keratin blot-binding.-IEF Isoelectric focusing.-IF Intermediate filament.-K Keratin.-MALDI-MS Matrix-assisted laser desorption/ionization moss spectrometry.-NEPHGE Non-equilibrium pH-gradient electrophoresis.-PVDF Polyvinylidene fluoride.-SDS Sodium dodecyl sulfate.-2D-PAGE Two-dimensional polyacrylamide gel electrophoresis.
Spectophotometric quantification is a very exact method of quantifying the content of nucleic aci... more Spectophotometric quantification is a very exact method of quantifying the content of nucleic acids in any sample. The purity of the sample can be simultaneously checked very easily. Keywords: DNA; RNA; spectrophotometric quantification
Background: Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indi... more Background: Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indirect development, either via a swimming-or a creeping-type larva and still debated phylogenetic position within Lophotrochozoa. Recent morphological and neuro-anatomical studies on the creeping-type larva support a close relationship of Entoprocta and Mollusca, with a number of shared apomorphies including a tetraneurous nervous system and a complex serotonin-expressing apical organ. However, many morphological traits of entoproct larvae, in particular of the putative basal creeping-type larva, remain elusive. Results: Applying fluorescent markers and 3D modeling, we found that this larval type has the most complex musculature hitherto described for any lophotrochozoan larva. The muscle systems identified include numerous novel and most likely creeping-type larva-specific structures such as frontal organ retractors, several other muscle fibers originating from the frontal organ, and longitudinal prototroch muscles. Interestingly, we found distinct muscle sets that are also present in several mollusks. These include paired sets of dorso-ventral muscles that intercross ventrally above the foot sole and a paired enrolling muscle that is distinct from the musculature of the body wall. Conclusion: Our data add further morphological support for an entoproct-mollusk relationship (Tetraneuralia) and strongly argue for the presence of an enrolling musculature as well as seriality (but not segmentation) in the last common tetraneuralian ancestor. The evolutionary driving forces that have led to the emergence of the extraordinarily complex muscular architecture in this short-lived, non-feeding entoproct larval type remain unknown, as are the processes that give rise to the highly different and much simpler muscular bodyplan of the adult entoproct during metamorphosis.
Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiolog... more Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon-intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation.
The analytical procedure for the quantification of DNA and RNA described is the quantitative visu... more The analytical procedure for the quantification of DNA and RNA described is the quantitative visual comparison of an unknown sample with known standards. This is quick and easy to do, suitable for amounts of sample as small as 10 ng. Keywords: DNA; RNA; rapid quantification
Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most mo... more Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-) decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins.
Journal of phylogenetics & evolutionary biology, 2018
Hox genes are highly conserved developmental genes involved in the patterning of the anterior-pos... more Hox genes are highly conserved developmental genes involved in the patterning of the anterior-posterior axis of nearly all metazoan animals. While Hox genes have been characterized for many bilaterians, several cryptic taxa, often comprising microscopic specimens, have hitherto been neglected. We here present the first combined transcriptomic and genomic Hox gene study for Entoprocta (=Kamptozoa), a phylum of microscopic, sessile, tentacle-bearing animals with unresolved phylogenetic affinities. We identified 10 of the 11 Hox genes commonly found in other lophotrochozoans. The analyses of transcriptomic data of different developmental stages of three species (regenerating stages of the colonial species Pedicellina cernua, budding stages of the solitary species Loxosomella vivipara and embryos of the solitary species Loxosomella murmanica/atkinsae) yielded the Hox genes Labial, Hox3, Lox5, and Post2 in all species. Pb and Dfd were only found being expressed in the colonial species P. cernua. Lox4 was uniquely expressed in the solitary species L. vivipara and L. murmanica/atkinsae. Other homeobox genes belonging to the ANTP-class genes, e.g., ParaHox and NK-like genes, were also found. Thus, in addition to newly identified Hox genes (PceLox2-like & LviPost2-like), Entoporocta show the typical lophotrochozoan Hox pattern besides the loss of the posterior class Hox gene Post1.
By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibo... more By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodetes. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of-M r 180000 and a structurally nonrelated cytoplasmic protein of M r 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.
Molecular Phylogenetics and Evolution, Feb 1, 2011
The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of... more The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda-Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia-Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated divergence time of Spirulida and Sepiida, some 150 ± 30 million years ago, can be deemed reliable.
Kamptozoa, also known as Entoprocta, are small aquatic filter-feeders that belong to the Lophotro... more Kamptozoa, also known as Entoprocta, are small aquatic filter-feeders that belong to the Lophotrochozoan superphylum, which also contains other acoelomate phyla including Annelida, Nemertea, and Mollusca. The study of Kamptozoa is thus of great interest to understand the early Lophotrochozoan evolution. Moreover, many kamptozoans have been shown to possess great regeneration capacities, including whole-body regeneration. In addition, and in particular for colonial cosmopolitan species such as Barentsia benedeni, kamptozoans are highly suitable as laboratory model organisms because of their simple culture, low space requirements, transparency and rapid life cycle. This chapter provides a brief introduction into field collection, culturing techniques for both the animals as well as the algae required for their feeding, fixation, staining, and sequencing.
Additional file 7. Movie files. Distribution of splice sites positions in a 3D model of a hemocya... more Additional file 7. Movie files. Distribution of splice sites positions in a 3D model of a hemocyanin FU.
Journal of Cancer Research and Clinical Oncology, Oct 1, 2001
Keyhole limpet hemocyanin (KLH) is a wellestablished immune stimulant and hapten carrier, and Hal... more Keyhole limpet hemocyanin (KLH) is a wellestablished immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1, KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture,. amino acid sequence, gene structure, and recombinant production of KLH and HtH. Abbreviations FU functional unit • HtH Haliotis tuberculata hemocyanin • KLH keyhole limpet hemocyanin • OdH Octopus dofleini hemocyanin R4
To cite: Lieb B, Schmitt P. Randomised double-blind placebo-controlled intervention study on the ... more To cite: Lieb B, Schmitt P. Randomised double-blind placebo-controlled intervention study on the nutritional efficacy of a food for special medical purposes (FSMP) and a dietary supplement in reducing the symptoms of veisalgia.
Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canon... more Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canonical 110 kDa form of somatic cells (Richard and Bogenhagen, Dev. Biol. 146: 4-11, 1991). By immunofluorescence microscopy using variant-specific antibodies we show that this high molecular weight form is associated with lampbrush chromosome loops and the inner regions of the amplified nucleoli. Inhibition of topo I-activity by either Camptothecin-treatment or microinjection of neutralizing antibodies resulted in loop retraction and the condensation of chromosomes and amplified nucleoli. These data indicate that the oocyte-specific 165 kDa form of topo I is involved in transcriptional processes mediated by RNA polymerase I and II and is therefore functionally equivalent to the somatic cell 110 kDa counterpart.
Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastro... more Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the ''slit apparatus'', local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20620 nm. In blueblooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposedthough not proven-that in the rare red-blooded snail species they might synthesize and secrete the hemoglobin. However, the cellular secretion pathway for respiratory proteins, and the functional role(s) of the enigmatic rhogocyte slit apparatus are still unclear. Additional functions for rhogocytes have been proposed, notably a role in protein uptake and degradation, and in heavy metal detoxification. Here we provide new structural and functional information on the rhogocytes of the redblooded freshwater snail Biomphalaria glabrata. By in situ hybridization of mantle tissues, we prove that rhogocytes indeed synthesize hemoglobin. By electron tomography, the first three dimensional (3D) reconstructions of the slit apparatus are provided, showing detail of highly dense material in the cytoplasmic bars close to the slits. By immunogold labelling, we collected evidence that a major component of this material is actin. By genome databank mining, the complete sequence of a B. glabrata nephrin was obtained, and localized to the rhogocytes by immunofluorescence microscopy. The presence of both proteins fit the ultrastructure-based hypothesis that rhogocytes are related to mammalian podocytes and insect nephrocytes. Reactions of the rhogocytes to deprivation of food and cadmium toxification are also documented, and a possible secretion pathway of newly synthesized respiratory proteins through the slit apparatus is discussed.
We have studied individual keratins of an elasmobrancb, the shark Scyliorhinus stellaris (the les... more We have studied individual keratins of an elasmobrancb, the shark Scyliorhinus stellaris (the lesser-spotted dogfish). From various shark tissues, notably skin and stomach, cytoskeletal proteins were isolated and then separated by two-dimensional polyacrylamide gel electrophoresis. Using complementary keratin blot-binding assays and immunoblotting, among these proteins we identified a variety of type I and type II keratins. According to their tissue-specific expression, we distinguished Is and lIs keratins from IE and lIE keratins ("S" and "E" from "simple epitbelial" and "epidermal", respectively). Guinea pig antibodies which in immunoblots specifically labeled the entire range of identified shark keratins, and a monoclonal antibody specific for IE keratins were used for immunoOuorescence microscopy of a broad range of shark tissues. These experiments demonstrated that in this shark, keratin expression is largely restricted to epithelia and-in contrast to the situation in teleost fishes-is lacking in mesenchymally derived cells and tissues. Peptide mass mapping of the major electrophoretically separated shark keratin spots revealed that the identified Is, lIs and lIE polypeptides are modifications of a single genuine keratin, respectively, wbereas there are two different IE keratins. It, therefore, appears that in this shark most (if not all) of the keratin cytoskeleton is constituted by only five different gene products (each present in various modifications): a heterologous pair of "S" and three different "E" keratins. We sequenced three of them (Is, lIs and lIE) via eDNA Abbreviations. CKBB Complementary keratin blot-binding.-IEF Isoelectric focusing.-IF Intermediate filament.-K Keratin.-MALDI-MS Matrix-assisted laser desorption/ionization moss spectrometry.-NEPHGE Non-equilibrium pH-gradient electrophoresis.-PVDF Polyvinylidene fluoride.-SDS Sodium dodecyl sulfate.-2D-PAGE Two-dimensional polyacrylamide gel electrophoresis.
Spectophotometric quantification is a very exact method of quantifying the content of nucleic aci... more Spectophotometric quantification is a very exact method of quantifying the content of nucleic acids in any sample. The purity of the sample can be simultaneously checked very easily. Keywords: DNA; RNA; spectrophotometric quantification
Background: Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indi... more Background: Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indirect development, either via a swimming-or a creeping-type larva and still debated phylogenetic position within Lophotrochozoa. Recent morphological and neuro-anatomical studies on the creeping-type larva support a close relationship of Entoprocta and Mollusca, with a number of shared apomorphies including a tetraneurous nervous system and a complex serotonin-expressing apical organ. However, many morphological traits of entoproct larvae, in particular of the putative basal creeping-type larva, remain elusive. Results: Applying fluorescent markers and 3D modeling, we found that this larval type has the most complex musculature hitherto described for any lophotrochozoan larva. The muscle systems identified include numerous novel and most likely creeping-type larva-specific structures such as frontal organ retractors, several other muscle fibers originating from the frontal organ, and longitudinal prototroch muscles. Interestingly, we found distinct muscle sets that are also present in several mollusks. These include paired sets of dorso-ventral muscles that intercross ventrally above the foot sole and a paired enrolling muscle that is distinct from the musculature of the body wall. Conclusion: Our data add further morphological support for an entoproct-mollusk relationship (Tetraneuralia) and strongly argue for the presence of an enrolling musculature as well as seriality (but not segmentation) in the last common tetraneuralian ancestor. The evolutionary driving forces that have led to the emergence of the extraordinarily complex muscular architecture in this short-lived, non-feeding entoproct larval type remain unknown, as are the processes that give rise to the highly different and much simpler muscular bodyplan of the adult entoproct during metamorphosis.
Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiolog... more Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon-intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation.
The analytical procedure for the quantification of DNA and RNA described is the quantitative visu... more The analytical procedure for the quantification of DNA and RNA described is the quantitative visual comparison of an unknown sample with known standards. This is quick and easy to do, suitable for amounts of sample as small as 10 ng. Keywords: DNA; RNA; rapid quantification
Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most mo... more Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-) decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins.
Journal of phylogenetics & evolutionary biology, 2018
Hox genes are highly conserved developmental genes involved in the patterning of the anterior-pos... more Hox genes are highly conserved developmental genes involved in the patterning of the anterior-posterior axis of nearly all metazoan animals. While Hox genes have been characterized for many bilaterians, several cryptic taxa, often comprising microscopic specimens, have hitherto been neglected. We here present the first combined transcriptomic and genomic Hox gene study for Entoprocta (=Kamptozoa), a phylum of microscopic, sessile, tentacle-bearing animals with unresolved phylogenetic affinities. We identified 10 of the 11 Hox genes commonly found in other lophotrochozoans. The analyses of transcriptomic data of different developmental stages of three species (regenerating stages of the colonial species Pedicellina cernua, budding stages of the solitary species Loxosomella vivipara and embryos of the solitary species Loxosomella murmanica/atkinsae) yielded the Hox genes Labial, Hox3, Lox5, and Post2 in all species. Pb and Dfd were only found being expressed in the colonial species P. cernua. Lox4 was uniquely expressed in the solitary species L. vivipara and L. murmanica/atkinsae. Other homeobox genes belonging to the ANTP-class genes, e.g., ParaHox and NK-like genes, were also found. Thus, in addition to newly identified Hox genes (PceLox2-like & LviPost2-like), Entoporocta show the typical lophotrochozoan Hox pattern besides the loss of the posterior class Hox gene Post1.
By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibo... more By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodetes. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of-M r 180000 and a structurally nonrelated cytoplasmic protein of M r 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.
Molecular Phylogenetics and Evolution, Feb 1, 2011
The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of... more The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda-Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia-Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated divergence time of Spirulida and Sepiida, some 150 ± 30 million years ago, can be deemed reliable.
Kamptozoa, also known as Entoprocta, are small aquatic filter-feeders that belong to the Lophotro... more Kamptozoa, also known as Entoprocta, are small aquatic filter-feeders that belong to the Lophotrochozoan superphylum, which also contains other acoelomate phyla including Annelida, Nemertea, and Mollusca. The study of Kamptozoa is thus of great interest to understand the early Lophotrochozoan evolution. Moreover, many kamptozoans have been shown to possess great regeneration capacities, including whole-body regeneration. In addition, and in particular for colonial cosmopolitan species such as Barentsia benedeni, kamptozoans are highly suitable as laboratory model organisms because of their simple culture, low space requirements, transparency and rapid life cycle. This chapter provides a brief introduction into field collection, culturing techniques for both the animals as well as the algae required for their feeding, fixation, staining, and sequencing.
Additional file 7. Movie files. Distribution of splice sites positions in a 3D model of a hemocya... more Additional file 7. Movie files. Distribution of splice sites positions in a 3D model of a hemocyanin FU.
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