Wolfgang Gebauer

Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to the acute fish test

Abstract The present study compares two possible alternative methods to replace the acute fish test. Fertilized eggs of zebrafish, Brachydanio rerio were used to investigate the acute toxicity of chemicals. Different toxicological endpoints such as coagulation of the eggs, development of gastrulation, number of somites, development of organs, circulation, heartbeat, otolithanlage and pigmentation have been examined during the embryonic development of zebrafish within the first 48 h. Differences to the normal development were measured by these endpoints. Chemicals have been examined in a preliminary approach with respect to their cytotoxicity on the RTG-2 cells (Rainbow Trout Gonad). The vitality of cells was measured by Neutral Red-and MTT assay. Urea, sodium chloride, 2-nitroanisole, 3,4-dichloroaniline, 4-nitrophenol, phenol, 2,4-dinitrophenol, dinitro-ortho-cresole, carbaryl and malathion were used as compounds with different modes of actions. The results of the cytotoxicity tests with Neutral Red uptake (NR50) and MTT (MTT50) have been compared with data of zebrafish, Brachydanio rerio. We could show that the embryo test in most cases is more sensitive than the acute toxicity test with adult zebrafish and in all cases is more sensitive than the RTG-2 cell test. Therefore, considering the wide range of possibilities the embryo of zebrafish offers, we think that the embryo test is a very good candidate to replace the acute fish test. Whether the cytotoxicity test with RTG-2 cells is also an alternative requires further, more detailed studies.

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, andHaliotis 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 limpetMegathura crenulata and the abaloneH. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1, KLH2, HIM, 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.

cDNA Sequence, Protein Structure, and Evolution of the Single Hemocyanin from Aplysia californica, an Opisthobranch Gastropod

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5′UTR of 77 bp, a 3′UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (Mr ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (Mr ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373 ± 47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343 ± 43 million years ago.

Identification, structure, and properties of hemocyanins from Diplopod myriapoda.

Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin ofSpirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 × 6) native molecule. It has a high oxygen affinity (P 50 = 4.7 torr) but low cooperativity (h = 1.3 ± 0.2).Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon, Scutigera coleoptrata (Chilopoda), indicating a rather conservative architecture of the myriapod hemocyanins. Western blotting demonstrates shared epitopes of Spirostreptus hemocyanin with both chelicerate and crustacean hemocyanins, confirming its identity as an arthropod hemocyanin.

Keyhole limpet haemocyanin: negative staining in the presence of trehalose

Abstract Samples of unpurified and purified haemocyanin from the giant keyhole limpet Megathura crenulata have been studied by transmission electron microscopy (TEM) using mixtures of trehalose with the negative stains, uranyl acetate and ammonium molybdate. Trehalose is a known protein preservative during air and freeze drying, UV irradiation and high temperatures, and therefore offers the possibility of protecting proteins during the drying of negatively-stained specimens and their subsequent electron microscopical study. Evidence is presented that trehalose possesses satisfactory stability within the electron beam during conventional room temperature, negative-staining studies. The combination of negative stain and trehalose generates a deeper layer of amorphous stain around and within the keyhole limpet haemocyanin (KLH) di- and multidecamers, thereby increasing protein mobility and the generation of a range of tilted images, alongside the more usually observed end-on and side-on image projections. The concentration of uranyl acetate and ammonium molybdate has been increased to 4 and 5%, respectively, in the presence of 1% trehalose, in order to generate satisfactory image contrast. This is because of the reduction by trehalose of the net mass thickness of the dried negative stain-carbohydrate mixture compared to for an equivalent thickness of stain alone. The study of these specimens at low temperatures (−175°C) offers the possibility of superior structural preservation.

Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod Haliotis tuberculata.

Abstract. Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rhogocytes, which confirmed that they are the site of hemocyanin biosynthesis in this gastropod. A possible path of hemocyanin release into the hemolymph is discussed. Also in the vetigastropod Megathura crenulata, many rhogocytes could be detected. However, they lacked hemocyanin molecules which, together with published data, indicates a seasonal expression of hemocyanin in this animal.

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel® followed by separation of KLH1 and KLH2

Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassociated mixture as homo-oligomers/polymers and can be separated by selective dissociation of the KLH2 by treatment with 1% ammonium molybdate-0.2% PEG at pH 5.7, followed by gel filtration chromatography in this solution. In addition to discrete elution peaks containing didecameric KLH1 and dissociated subunits of KLH2, a leading peak contains a tubular/polymeric form of KLH1, not previously described. Under negative staining in conditions designed specifically for the creation of 2-dimensional crystals on mica (the negative staining-carbon film procedure), this tubular form of KLH1 can be transformed into a larger diameter multidecameric form, again not previously described for KLH1. The purified KLH2 peak is indistinguishable from subunit material prepared from living animals. This, Immucothel appears to provide a standardized source of subunits suitable for biochemical and structural studies on the two types of KLH.

Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2

Abstract Keyhole limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the form of didecamers and small clusters of PEG at pH 5.9 (pH 6.2 at 4°C). Assessment of the achieved separation of KLH1 and KLH2 was monitored by gel electrophoresis under native conditions and by electron microscopy of negatively stained specimens. Integrity of the multi-domain KLH1 and KLH2 subunits was assessed by SDS-PAGE. The significance of this separation for the overall understanding of the two types of KLH is that it provides for the first time purified KLH1 didecamers. Reassociation of the pH 5.9 dissociated KLH2 was achieved by prolonged dialysis against calcium-supplemented stabilizing buffer at pH 7.2 and resulted in multidecamers and slightly smaller diameter helical tubes.

Red blood with blue-blood ancestry: Intriguing structure of a snail hemoglobin

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin “plug” domain with three cysteines for subunit dimerization (total Mr ≈ 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical images (3 nm resolution); it suggests a 3 × 2-mer quaternary structure (Mr ≈ 1.43 MDa). Moreover, we identified a previously undescribed rosette-like hemolymph protein that has been mistaken for hemoglobin. We also detected expression of an incomplete hemocyanin as trace component. The combined data show that B. glabrata hemoglobin evolved from pulmonate myoglobin, possibly to replace a less-efficient hemocyanin, and reveals a surprisingly simple evolutionary mechanism to create a high molecular mass respiratory protein from 78 similar globin domains.

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.