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.

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.

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.

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.

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.

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.

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.

Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods.

Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha-tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.

Suppression of the modulatory effects of the antileukemic and anti-human immunodeficiency virus compound avarol on gene expression by tryptophan.

The amino acid L-tryptophan is known to be a modulator of many processes of cell metabolism. In this contribution we show that L-tryptophan interferes with some biological effects of the antileukemic and anti-human immunodeficiency virus agent avarol, possibly by different mechanisms. Avarol has been shown to be able to modulate posttranscriptional events of mRNA synthesis, resulting in an increase of the base-sequence complexities of the nonabundant and rare mRNA classes. Here it is demonstrated that this change in mRNA abundancy distribution is accompanied by an increase in the level of some specific, low abundant mRNAs (ras and c-myc). Addition of L-tryptophan was found to abolish avarol-caused gene relaxation in L1210 mouse leukemia cells. In addition, L-tryptophan suppressed the induction of gamma-interferon mRNA production in human peripheral blood lymphocytes. At the level of DNA, L-tryptophan inhibited the production of strand breaks by cytotoxic avarol concentrations in Friend erythroleukemia cells in vitro. Moreover, it competed with avarol for binding to the nuclear envelope binding site; this effect was not shown by other amino acids.

Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo.

Avarone and avarol are novel cytostatic agents which have potent antileukemic activity both in vitro and in vivo (mice). Cell culture experiments revealed that the cytostatic activity of these two compounds on L5178Y mouse lymphoma cells was 13- to 14-fold higher than that determined for HeLa cells and 40- to 43-fold higher than that for human melanoma cells. Nontumor cells (human fibroblasts and human gingival cells) were highly resistant against the two compounds. The inhibitory potency of avarone on L5178Y cells (50% inhibitory concentration, 0.62 microM) was significantly higher than the avarol activity (50% inhibitory concentration, 0.93 microM). Modification of the molecule at the quinone ring or the double bond in the terpenoid skeleton resulted in a significant loss of activity. In vivo studies with L5178Y cells in the ascites of mice confirmed the strong antileukemic effect determined in vitro. At doses of 10 mg/kg given i.p. once daily for 5 days to mice bearing approximately 10(8) leukemia cells, avarone was found to be curative in about 70% of the mice (20% for avarol). The optimal daily i.p. dose of avarone increased life span over controls by 146% when treatment was begun 1 day after tumor implantation and by 87% when treatment was delayed until day 8. Avarol, although active, was less effective. Based on the determined log10 kill values, avarone can be classified as a highly active and avarol as a markedly active cytostatic agent. The efficacy of the two compounds is also emphasized by the therapeutic index of 11.7 for avarone and of 4.5 for avarol. The two agents were determined not to be either direct mutagens or premutagens in the Ames test.

Avarol-induced DNA strand breakage in vitro and in Friend erythroleukemia cells.

The hydroquinone-containing cytostatic compound avarol inhibits predominantly growth of those cell lines which have a low level of superoxide dismutase. The substrate of this enzyme, the superoxide anion, was found to be formed during the in vitro oxidation reaction of avarol to its semiquinone radical in the presence of oxygen. Under the same incubation conditions plasmid DNA (pBR322) was converted from the fully supercoiled circular form mainly to the nicked circular form, indicating that the compound causes primarily single-strand breaks. Using Friend erythroleukemia cells (FLC) it was found that avarol induces a dose-dependent DNA damage; the maximum number of DNA strand breaks was observed at 5 h after addition of the compound to the cells. Removal of avarol resulted in a rapid DNA rejoining with biphasic repair kinetics [first half-time, 8 min (90% of the breaks) and a second half-time, 40 min (10% of the breaks)]. When the degree of avarol-induced DNA damage in FLC was compared with the drug-caused inhibition of cell growth a close correlation was established. Avarol displayed no effect on dimethyl sulfoxide-induced erythrodifferentiation of FLC as determined by the benzidine reaction and by dot blot hybridization experiments. From incubation studies of FLC with [3H]avarol no hint was obtained for the formation of an adduct between DNA and the compound. The subcellular distribution of [3H]avarol was studied in liver cells after i.v. application of the compound. The predominant amount of the compound was present in the cytosolic fraction; little avarol was associated with plasma membranes, nuclei, and mitochondria. Using (a) oxidative phosphorylation and (b) oxygen uptake as parameters for mitochondria function, no effect of the compound on the activity of this organelle was determined. These results suggest that avarol forms superoxide anions (and in consequence possibly also hydroxyl radicals) especially in those cells which have low levels of superoxide dismutase. Moreover, evidence is provided that the active oxygen species cause DNA damage resulting in the observed cytotoxic effect.

Inhibition of nuclear envelope nucleoside triphosphatase-regulated nucleocytoplasmic messenger RNA translocation by 9-beta-D-arabinofuranosyladenine 5'-triphosphate in rodent cells.

Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid [poly(A)] tail of messenger RNA (Bernd, A., Schröder, H. C., Zahn, R. K., and Müller, W. E. G. Eur. J. Biochem., 129: 43-49, 1982). RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei is strongly reduced if 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) is present in the transport medium. Half-maximal inhibition of RNA efflux occurs with 120 microM ara-ATP. Most likely, the inhibitory effect of ara-ATP is caused by inhibition of nuclear envelope nucleoside triphosphatase; this enzyme was found to be highly sensitive to inhibition by this antibiotic. The inhibition type of the nucleoside triphosphatase of rat liver nuclear ghosts is competitive with respect to ATP; the Ki:Km ratio was determined to be 0.27. Besides nucleoside triphosphatase, nuclear envelopes contain a protein phosphokinase modulating the affinity of pore complex laminae to poly(A). This enzyme was also found to be strongly inhibited by ara-ATP in a competitive way with respect to ATP (Ki:Km, 0.056) and could therefore also contribute to the overall inhibition of RNA transport. The polyadenylation of endogenous RNA by poly(A) polymerase(s) in intact rat liver nuclei as well as in nuclear matrices isolated from the same source was found to be markedly suppressed in the presence of ara-ATP. The inhibitions of both poly(A) polymerase activities (contained in whole nuclei or nuclear matrix bound) are of the competitive type with respect to ATP. In in vitro assays, nuclear envelope nucleoside triphosphatase is inhibited by microtubule protein. Of the 2 ATP-dependent enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and adenosine triphosphatase), only the kinase was slightly affected by ara-ATP. Cellular uptake of adenosine 5'-monophosphate and perhaps 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is facilitated by a cellular membrane-bound 5'-nucleotidase. Our studies revealed that neither cleavage of ara-AMP nor inhibition of the enzyme activity by ara-AMP occurs. 9-beta-D-Arabinofuranosyladenine and ara-AMP represent neither direct mutagens nor premutagens as determined by the Salmonella-mammalian microsome mutagenicity test.

Modulation of myb gene expression in sponges by retinoic acid.

We demonstrate that the cells of the sponge Geodia cydonium are equipped with the basic elements required for a retinoic acid (RA)-dependent response pathway; RA was identified and quantitated, the cellular RA-binding protein (CRABP) was detected and the nuclear RA receptor (RAR) was found. In the isolated cell system the level of CRABP, but not of RAR, is strongly induced after incubating the cells for 10h with the homologous aggregation factor. In induced cells incubation with 0.3 microM RA results in a strong down-regulation of the c-myb (or c-myb-related) proto-oncogene (M(r) 63,000; mRNA 3.3 kb). We postulate that this pathway is also functionally active and that RA acts as a natural morphogen.

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.

Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells.

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.

Purification and characterization of two exopolyphosphatases from the marine sponge Tethya lyncurium.

Two exopolyphosphatases (exopolyphosphatase I and II; EC 3.6.1.11) which release orthophosphate from inorganic polyphosphates have been detected and purified for the first time from a marine sponge. Tethya lyncurium. Exopolyphosphatase I has a molecular mass of 45 kDa, a pH optimum of 5.0 and does not require divalent cations for activity, while exopolyphosphatase II has a molecular mass of 70 kDa, a pH optimum of 7.5 and displays optimal activity in the presence of Mg2+ ions. Final purification of the enzymes could be achieved by affinity chromatography on polyphosphate-modified zirconia. The mode of action of both enzymes was found to be processive. Orthophosphate is the sole product formed by exopolyphosphatase I, while degradation of linear polyphosphates by exopolyphosphatase II occurs to pyrophosphate as end product, which is hydrolyzed, if at all, only very slowly. Significant amounts of polyphosphate (approximately 30 micrograms/g wet weight) were found to be present in the sponge organism. Polyphosphate is shown to inhibit the formation of ATP by adenylate kinase activity present in T. lyncurium extracts in a competitive manner. The inhibitory effect of long-chain polyphosphates was higher than that of short-chain polyphosphate, suggesting a potential role of polyphosphate metabolism in regulating intracellular concentrations of adenylate nucleotides.

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction.

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol Pi/min per 10(8) ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the 'carrier structure' for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.

Differential changes of nuclear-envelope-associated enzyme activities involved in nucleocytoplasmic mRNA transport in the developing rat brain and liver.

Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3'poly(A) tail of poly(A)+ mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A)- mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A)- mRNA unique in the brain.

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.

Accumulation of transcripts coding for prion protein in human astrocytes during infection with human immunodeficiency virus.

The abnormal isoforms of the normal cellular prion protein (PrP), also termed Scrapie-associated fibril protein, are assumed to be one causative factor of spongiform encephalopathies. The mRNA of PrP contains stem-loop structures which are very similar to the human immunodeficiency virus-1 (HIV-1) cis-acting sequence TAR within the LTR; both structures contain the pentanucleotide CUGGG in the loop, and the uridine- and adenine-bulge in the stem. In this study, using purified HIV-encoded trans-activator, Tat, and HIV-1 TAR-RNA or PrP-mRNA containing the stem-loop structure, we demonstrate by use of gel-retardation and filter binding assays that Tat binds to TAR- and PrP-RNA with the dissociation constants of 2.9 or 37.0 nM, respectively, at a molar ratio of 0.7 mol of Tat to 1 mol of RNA fragment. The Tat-RNA (TAR or PrP) complexes bind to protein(s) in the nuclear matrix, isolated from human astrocytes (glial fibrillary acidic protein positive brain cells). Infection of astrocytes with HIV-1 resulted in an increased level of PrP mRNA. The data presented led us to assume that certain sequences in the PrP mRNA might be targets for proteins acting in trans.