Polyphosphate (PolyP) for alveolar cleft repair: study protocol for a pilot randomized controlled trial.

OBJECTIVE: Bone grafting is an important surgical procedure to restore missing bone in patients with alveolar cleft lip/palate, aiming to stabilize either sides of the maxillary segments by inducing new bone formation, and in bilateral cleft cases also to stabilize the pre-maxilla. Polyphosphate (PolyP), a physiological polymer composed of orthophosphate units linked together with high-energy phosphate bonds, is a naturally existing compound in platelets which, when complexed with calcium as Ca-polyP microparticles (Ca-polyP MPs), was proven to have osteoinductive properties in preclinical studies. AIM: To evaluate the feasibility, safety, and osteoinductivity of Ca-polyP MPs as a bone-inducing graft material in humans. METHODS: This prospective non-blinded first-in-man clinical pilot study shall consist of 8 alveolar cleft patients of 13 years or older to evaluate the feasibility and safety of Ca-PolyP MPs as a bone-inducing graft material. Patients will receive Ca-polyP graft material only or Ca-polyP in combination with biphasic calcium phosphate (BCP) as a bone substitute carrier. During the trial, the participants will be investigated closely for safety parameters using radiographic imaging, regular blood tests, and physical examinations. After 6 months, a hollow drill will be used to prepare the implantation site to obtain a biopsy. The radiographic imaging will be used for clinical evaluation; the biopsy will be processed for histological/histomorphometric evaluation of bone formation. DISCUSSION: This is the first-in-man study evaluating the safety and feasibility of the polyP as well as the potential regenerative capacity of polyP using an alveolar cleft model. TRIAL REGISTRATION: Indonesian Trial Registry INA-EW74C1N . Registered on 12 June 2020.

Artificial cartilage bio-matrix formed of hyaluronic acid and Mg2+-polyphosphate.

Here we show that inorganic polyphosphate (polyP), a polyanionic metabolic regulator consisting of multiple phosphate residues linked by energy-rich phosphoanhydride bonds, is present in the synovial fluid. In a biomimetic approach, to enhance cartilage synthesis and regeneration, we prepared amorphous polyP microparticles with Mg2+ as counterions. The particles were characterised by X-ray diffraction (XRD), energy-dispersive X-ray (EDX) and Fourier transformed infrared spectroscopic (FTIR) analyses. Similar particles were obtained after addition of Mg2+ ions to a solution containing hyaluronic acid, as a major component of the synovial fluid, and soluble Na-polyP. The viscous paste-like material formed, composed of globular microparticles with diameter of 400 nm, strongly promoted the adhesion of chondrocytes and caused a significant upregulation of the expression of the genes encoding collagen type 3A1, as a marker for chondrocyte differentiation, and SOX9, a transcription factor that regulates chondrocyte differentiation and proliferation. The expression level of the collagen type 3A1 gene was also enhanced by exposure of chondrocytes to synovial fluid that was found to contain polyP with a size of about 80 phosphate residues. This stimulatory effect was abolished after pre-incubation of the synovial fluid with the polyP degrading alkaline phosphatase. We propose a strategy for treatment of joint dysfunctions caused by osteoarthritis based on the application of amorphous Mg2+-polyP microparticles thatprevent calcium crystal formation in the synovial fluid using scavenging Ca2+ ions (Mg2+/Ca2+ exchange) and enhance chondrocyte function after binding of the Ca2+-polyP to hyaluronic acid at the cartilage surface.

Morphogenetically active scaffold for osteochondral repair (polyphosphate/alginate/N,O-carboxymethyl chitosan).

Here we describe a novel bioinspired hydrogel material that can be hardened with calcium ions to yield a scaffold material with viscoelastic properties matching those of cartilage. This material consists of a negatively charged biopolymer triplet, composed of morphogenetically active natural inorganic polyphosphate (polyP), along with the likewise biocompatible natural polymers N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The porosity of the hardened scaffold material obtained after calcium exposure can be adjusted by varying the pre-processing conditions. Various compression tests were applied to determine the local (nanoindentation) and bulk mechanical properties (tensile/compression test system for force measurements) of the N,O-CMC-polyP-alginate material. Determinations of the Young's modulus revealed that the stiffness of this comparably water rich (and mouldable) material increases during successive compression cycles to values measured for native cartilage. The material not only comprises viscoelastic properties suitable for a cartilage substitute material, but also displays morphogenetic activity. It upregulates the expression of genes encoding for collagen type II and aggrecan, the major proteoglycan within the articular cartilage, in human chondrocytes, and the expression of alkaline phosphatase in human bone-like SaOS-2 cells, as revealed in RT qPCR experiments. Further, we demonstrate that the new polyP-based material can be applied for manufacturing 3D solid models of cartilage bone such as of the tibial epiphyseal plate and the superior articular cartilage surface. Since the material is resorbable and enhances the activity of cells involved in regeneration of cartilage tissue, this material has the potential to be used for artificial articular cartilage implants.

In vivo ozone exposure does not increase DNA single-strand breaks in human peripheral lymphocytes.

In this randomized parallel study, we examined whether an acute ozone (O3) exposure leads to increased DNA strand breaks in human lymphocytes. The groups were exposed to 0.21 ppm O3 or filtered air for two hours. 30min and 4.5 h after exposure, DNA damage was determined in isolated lymphocytes using the Fast Micromethod. There was no detectable effect after O3 exposure. We conclude that an acute O3 exposure at the tested concentration does not lead to persistent DNA damage.

Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation.

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system, using SaOS-2 cells and RAW 264.7 cells, it is shown that in the presence of 10 µM silicate the number of RAW 264.7 cells in general, and the number of TRAP(+) RAW 264.7 cells in particular markedly decreases. The SaOS-2 cells retain their capacity of differential gene expression of OPG and RANKL in favor of OPG after exposure to silicate. It is concluded that after exposure of the cells to silicate a factor(s) is released from SaOS-2 cells that causes a significant inhibition of osteoclastogenesis of RAW 264.7 cells. It is assumed that it is an increased secretion of the cytokine OPG that is primarily involved in the reduction of the osteoclastogenesis of the RAW 264.7 cells. It is proposed that silicate might have the potential to stimulate osteogenesis in vivo and perhaps to ameliorate osteoporotic disorders.

Induction of apoptosis in mussel Mytilus galloprovincialis gills by model cytotoxic agents.

Apoptosis signaling pathway was investigated in the marine mussel Mytilus galloprovincialis exposed to various stressors. Analyses were performed in mussels exposed to two major pollutants of the aquatic environment: tributyltin and the water soluble fraction of diesel oil, for 1 h and animals were then maintained in sea water for a recovery period of 6 and 24 h. Apoptosis was evaluated at several levels of the cell signaling cascade by measuring Bcl-xS expression, caspase-3 activity and DNA damage (Fast micromethod(®) and TUNEL techniques). H(2)O(2) was used as a control of apoptosis induction for validation of the assays. Results showed an induction of Bcl-xS expression, a protein implicated in apoptosis, after 1 h exposure to all concentrations of chemicals. Moreover, in the same manner, apoptotic DNA damage was induced with all chemicals tested. Besides, caspase 3 activity was detected after 1 h exposure to low doses of TBT and diesel oil while the high concentrations induced this protein after 6 h. The achieved data were also correlated with our previous study, demonstrating an induction of the mitogen-activated protein kinase (MAPK) activity in the mussel M. galloprovincialis exposed to the same conditions. In conclusion, this study was one of the first characterizing the MAP kinase cell signaling pathway leading to apoptosis in the mussel M. galloprovincialis exposed to chemicals. It showed for the first time that the Bcl-xS protein was present in these mussels as in other species and played a role in apoptosis mediation. Moreover, the main originality of this work was that it showed that two apoptotic pathways might be present in the mussel: a caspase 3-dependent and a caspase 3-independent pathways.

MAP kinase cell signaling pathway as biomarker of environmental pollution in the sponge Suberites domuncula.

In the present study, we analyzed the effects of two major pollutants of the environment, tributyltin (TBT) and water-accommodated fraction (WAF) of diesel oil, on MAP kinase activation, apoptosis induction and DNA damage, in the marine sponge Suberites domuncula. Our results clearly demonstrated a differential activation of the MAPKs depending on the chemicals tested. TBT induced the activation of p38 and JNK while diesel oil enhanced activation of both ERK and p38. The activation of MAPKs was observed after 1 h exposure and 6 and 24 h of recovery in seawater. In addition, DNA fragmentation, assessed by two techniques, the Fast micromethod(®) and the TUNEL assay, was detected after sponges were treated with both chemicals. Moreover, the study of caspase 3/7 activity showed that apoptosis was induced and triggered with all concentrations of TBT but only at high diesel oil concentrations. After TBT exposure, a correlation was observed between JNK activation, caspase 3 activity and DNA damage while p38 activation followed the two latter parameters at high concentrations of diesel oil, suggesting that sponges enhanced a specific apoptotic pathway depending on the xenobiotic tested. This study demonstrated a high signal response by the sponge Suberites domuncula to the tested chemicals. Cell signaling pathway studies may thus be of use in water quality biomonitoring programs.

Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution.

Luciferase a light source for the silica-based optical waveguides (spicules) in the demosponge Suberites domuncula.

Two classes of sponges (animal phylum Porifera) possess a siliceous skeleton which is composed of spicules. Studying the optical fiber-mechanical properties of large spicules from hexactinellid sponges (> 5 cm) it was demonstrated that they are effective light-collecting optical fibers. Here, we report that the demosponge Suberites domuncula is provided with a biosensor system composed of the (organic) light producing luciferase and the (inorganic) light transducing silica spicules. The light transmission feature of these smaller spicules (200 microm) has been demonstrated and the ability of sponge tissue to generate light has been proven. Screening for a luciferase gene in S. domuncula was successful; the recombinant luciferase was prepared and shown to be bioactive. The luciferase protein is abundantly present in the close neighborhood of the spicules. The expression of the luciferase gene is under the control of light.

Monitoring chemical and physical stress using sea urchin immune cells.

Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stress. Only recently, coelomocytes have been used for pollution studies with the aim of introducing a new biosensor for detection of stress at both cellular and molecular levels, as sentinel of sea health. In this chapter, we briefly review the important features of these valuable cells and describe studies on their use in the laboratory and in the field for the assessment of chemical and physical pollution of the sea.

DNA damage and developmental defects after exposure to UV and heavy metals in sea urchin cells and embryos compared to other invertebrates.

The depletion of the stratospheric ozone layer and the resulting increase in hazardous ultraviolet-B (UV-B) radiation reaching the Earth are of major concern not only for terrestrial but also for aquatic organisms. UV-B is able to penetrate clear water to ecologically significant depths. This chapter deals with the effects of UV radiation on DNA integrity in marine benthic organisms, in particular sea urchins in comparison to other marine invertebrates (sponges and corals). These animals cannot escape the damaging effects of UV-B radiation and may be additionally exposed to pollution from natural or anthropogenic sources. Besides eggs and larvae that lack a protective epidermal layer and are particularly prone to the damaging effects of UV radiation, coelomocytes from the sea urchin Paracentrotus lividus were used as a "cellular sensor" to analyse the effects on DNA caused by UV-B, heavy metals (cadmium), and their combined actions. From our data we conclude that sea urchin coelomocytes as well as cells from other marine invertebrates are useful bioindicators of UV-B and heavy metal stress, responding to these stressors with different extents of DNA damage.

The chemokine networks in sponges: potential roles in morphogenesis, immunity and stem cell formation.

Porifera (sponges) are now well accepted as the phylum which branched off first from the common ancestor of all metazoans, the Urmetazoa. The transition to the Metazoa became possible because during this phase, cell-cell as well as cell-matrix adhesion molecules evolved which allowed the formation of a colonial stage of animals. The next prerequisite for the evolution to the Urmetazoa was the establishment of an effective immune system which, flanked by apoptosis, allowed the formation of a first level of individuation. In sponges (with the model Suberites domuncula and Geodia cydonium), the main mediators of the immune responses are the chemokines. Since sponges lack a vascular system and consequently blood cells (in the narrow sense), we have used the term chemokines (in a broad sense) to highlight that the complex network of intercellular mediators initiates besides differentiation processes also cell movement. In the present review, the cDNAs encoding the following chemokines were described and the roles of their deduced proteins during self-self and nonself recognition outlined: the allograft inflammatory factor, the glutathione peroxidase, the endothelial-monocyte-activating polypeptide, the pre-B-cell colony-enhancing factor and the myotrophin as well as an enzyme, the (2-5)A synthetase, which is involved in cytokine response in vertebrates. A further step required to reach the evolutionary step of the integrated stage of the Urmetazoa was the acquisition of a stem cell system. In this review, first markers for stem cells (mesenchymal stem cell-like protein) as well as for chemokines involved in the maintenance of stem cells (noggin and glia maturation factor) are described at the molecular level, and a first functional analysis is approached. Taken together, it is outlined that the chemokine network was essential for the establishment of metazoans, which evolved approximately 600 to 800 million years ago.

Sustainable production of bioactive compounds from sponges: primmorphs as bioreactors.

Sponges [phylum Porifera] are a rich source for the isolation of biologically active and pharmacologically valuable compounds with a high potential to become effective drugs for therapeutic use. However, until now, only one compound has been introduced into clinics because of the limited amounts of starting material available for extraction. To overcome this serious problem in line with the rules for a sustainable use of marine resources, the following routes can be pursued; first, chemical synthesis, second, cultivation of sponges in the sea (mariculture), third, growth of sponge specimens in a bioreactor, and fourth, cultivation of sponge cells in vitro in a bioreactor. The main efforts to follow the latter strategy have been undertaken with the marine sponge Suberites domuncula. This species produces compounds that affect neuronal cells, such as quinolinic acid, a well-known neurotoxin, and phospholipids. A sponge cell culture was established after finding that single sponge cells require cell-cell contact in order to retain their telomerase activity, one prerequisite for continuous cell proliferation. The sponge cell culture system, the primmorphs, comprises proliferating cells that have the potency to differentiate. While improving the medium it was found that, besides growth factors, certain ions (e.g. silicate and iron) are essential. In the presence of silicate several genes required for the formation of the extracellular matrix are expressed (silicatein, collagen and myotrophin). Fe3+ is essential for the synthesis of the spicules, and causes an increased expression of the ferritin-, septin- and scavenger receptor genes. Furthermore, high water current is required for growth and canal formation in the primmorphs. The primmorph system has already been successfully used for the production of pharmacologically useful, bioactive compounds, such as avarol or (2'-5')oligoadenylates. Future strategies to improve the sponge cell culture are discussed; these include the elucidation of those genes which control the proliferation phase and the morphogenesis phase, two developmental phases which the cells in primmorphs undergo. In addition, immortalization of sponge cells by transfection with genomic DNA appears to be a promising way, since recent studies underscore the applicability of this technique for sponges.

Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa).

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.

Suppression of allograft rejection in the sponge Suberites domuncula by FK506 and expression of genes encoding FK506-binding proteins in allografts.

Porifera (sponges) are, evolutionarily, the oldest metazoan phylum. Recent molecular data suggest that these animals possess molecules similar to and homologous with those of the innate and adaptive immune systems of higher Metazoa. Applying the biological system of parabiosis and the technique of differential display of mRNA, two cDNAs encoding putative FK506-binding proteins were isolated. FK506 is successfully used in clinics as a drug to prevent allograft rejection and is toxic to Suberites domuncula cells in vitro at doses above 100ng ml(-1). Autograft fusion of transplants from S. domuncula was not affected by FK506. Allograft non-fusion was not affected by FK506 at toxic doses; however, at the non-toxic dose of 20ng ml(-1), the allografts fused with each other. It is shown that at the attachment zone in untreated and (particularly drastic) in FK506-treated allografts, expression of the genes encoding the FK506-binding proteins is upregulated. These data indicate that the drug FK506 suppresses allograft rejection in S. domuncula, most probably via interaction with expression of the gene coding for the FK506-binding proteins.

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1).

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schröder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.

Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase.

Recent results revealed that inorganic polyphosphates (polyP), being energy-rich linear polymers of orthophosphate residues known from bacteria and yeast, also exist in higher eukaryotes. However, the enzymatic basis of their metabolism especially in mammalian cells is still uncertain. Here we demonstrate for the first time that alkaline phosphatase from calf intestine (CIAP) is able to cleave polyP molecules up to a chain length of about 800. The enzyme acts as an exopolyphosphatase degrading polyP in a processive manner. The pH optimum is in the alkaline range. Divalent cations are not required for catalytic activity but inhibit the degradation of polyP. The rate of hydrolysis of short-chain polyP by CIAP is comparable to that of the standard alkaline phosphatase (AP) substrate p-nitrophenyl phosphate. The specific activity of the enzyme decreases with increasing chain length of the polymer both in the alkaline and in the neutral pH range. The K(m) of the enzyme also decreases with increasing chain length. The mammalian tissue non-specific isoform of AP was not able to hydrolyze polyP under the conditions applied while the placental-type AP and the bacterial (Escherichia coli) AP displayed polyP-degrading activity.

Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene.

BACKGROUND: Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase. RESULTS: Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells). The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu. CONCLUSION: The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone) in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.

Determination of 14-3-3 protein levels in cerebrospinal fluid from Creutzfeldt-Jakob patients by a highly sensitive capture assay.

The level of 14-3-3gamma protein was determined in the cerebrospinal fluid (CSF) from patients with Creutzfeldt-Jakob disease (CJD) and non-CJD patients applying a new and fast microplate assay (14-3-3 protein capture assay), based on the binding to a peptide comprising a phosphorylated recognition motif of 14-3-3 protein. The levels of the gamma-isoform of 14-3-3 protein in CSF samples from CJD patients (n=41) were significantly higher than those observed in patients with non-CJD dementias (n=36) suggesting that this capture assay is a reliable method in the diagnosis of CJD. Since this assay allows the direct measurement of 14-3-3 protein in the CSF without prior concentration it is an easy and simple alternative to the conventionally applied immunoblotting procedures.

Receptor protein-tyrosine phosphatases: origin of domains (catalytic domain, Ig-related domain, fibronectin type III module) based on the sequence of the sponge Geodia cydonium.

Reversible tyrosine phosphorylation of proteins is one of the major regulatory physiological events in response to cell-cell- and cell-matrix contact in Metazoa. Previously it was documented that the tyrosine phosphorylating enzymes, the tyrosine kinases (TKs), are autapomorphic characters of Metazoa, including sponges. In this paper the tyrosine dephosphorylating enzymes, the protein-tyrosine phosphatases (PTPs), are studied which can be grouped into two subfamilies, the soluble PTPs and the receptor PTPs (RPTPs). PTPs are characterized by one PTPase domain which interestingly comprises sequence similarity to yeast PTPs. In contrast to the PTPs, the RPTPs - which have been found only in Metazoa - are provided with two PTPase domains. To study the evolution of the RPTPs the full-length size RPTP was cloned from the marine demosponge Geodia cydonium, the phylogenetic oldest metazoan taxon. The 3253 bp long sequence has a putative open reading frame coding for a 999 aa long RPTP which is characterized by two fibronectin (type III; FN-III) domains in the extracellular portion, one intracellular immunoglobulin (Ig)-related domain, and two PTPase domains. Phylogenetic analysis revealed that the sponge FN-III domains form the basis of the metazoan FN-III domain with the common metazoan ancestor. The Ig-related, typical metazoan, module is classified to the disulphide lacking Ig members and represents the phylogenetic earliest member of this group. The beta-sheet propensity was calculated and the characteristic amino acids are present in the seven beta-sheets. The analysis of the two PTPase domains of the sponge RPTP demonstrates that the first domain is closely related to the PTPase domains present in the soluble PTPs, while the second PTPase domain is only distantly related to them. By constructing a rooted phylogenetic cladogram it became overt that the duplication of the PTPase domains must have occurred already in yeast. This interesting finding indicates that two conserved PTPase domains originated from a common ancestor in yeast while the evolutionary novelties, the FN-III domains and the Ig-related module, were added during the transition to the Metazoa. Hence, the tyrosine dephosphorylating enzyme, RPTP, is an example for a modular protein which is composed of ancient modules (PTPase domain[s]) and two metazoan novelties, while the tyrosine phosphorylating enzymes, the TKs, evolved only in Metazoa.