0000000000179099
AUTHOR
Bernd Lorenz
Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species.
Abstract Flupirtine (KATADOLON®), known as a nonopiate centrally acting analgesic drug, was tested as to its potential to prevent apoptosis of human endothelial cells induced by reactive oxygen species (ROS). It was found that Flupirtine displayed no effect on viability and cell proliferation of human umbilical vein endothelial cells (HUVEC) up to a concentration of 10 μg/mL. Apoptosis, induced by ROS and generated by hypoxanthine/xanthine oxidase (EC 1.1.3.22) (HX/XOD) or t-butyl hydroperoxide, was reduced after preincubation with Flupirtine for 3 hr by 35% and 41%, respectively. The maximal cytoprotective effect against apoptosis was observed at a drug concentration of 1 to 3 μg/mL. Flow …
Inorganic Polyphosphate in Human Osteoblast-like Cells
Significant amounts of inorganic polyphosphates and of polyphosphate-degrading exopolyphosphatase activity were detected in human mandibular-derived osteoblast-like cells. The amount of both soluble and insoluble long-chain polyphosphate in unstimulated osteoblast-like cells was higher than in human gingival cells, erythrocytes, peripheral blood mononuclear cells, and human blood plasma. The cellular content of polyphosphate in osteoblast-like cells strongly decreased after a combined treatment of the cells with the stimulators of osteoblast proliferation and differentiation, dexamethasone, beta-glycerophosphate, epidermal growth factor, and ascorbic acid. The amount of soluble long-chain p…
Regulation of motility of cells from marine sponges by calcium ions
Sponges are known not to contain muscle and nerve cells. Since sponge cells are characterized by high motility we determined the effect of intracellular calcium ion concentration ([Ca2+]i) on their motility. Addition of the Ca2+ ionophore ionomycin to dissociated cells from the marine sponge Suberites domuncula caused in Ca(2+)-containing artificial seawater (ASW) an increase in motility from 0.2 micron/min (absence of the ionophore) to 3.7 microns/min (presence of ionomycin). When the experiments were performed in Ca(2+)-free medium, no effect of ionomycin could be observed. In parallel experiments the changes of [Ca2+]i using the dye Fura-2 were measured. The experiments revealed that ion…
Purification and characterization of two exopolyphosphatases from the marine sponge Tethya lyncurium
Abstract 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 Mg 2+ 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 p…
Inorganic polyphosphates in the developing freshwater spongeEphydatia muelleri: Effect of stress by polluted waters
Relatively high amounts of inorganic polyphosphates (approximately 55 μg of polyphosphate/g of wet weight) were found in the freshwater sponge Ephydatia muelleri, particularly in the gemmules (260 μg/g). Here we report that the polyphosphate content of this sponge changes during development and in response to adverse environmental conditions. Germination and hatching of gemmules of E. muelleri is accompanied by a strong decrease (by 94% at day 2) in polyphosphate level and a rise in exopolyphosphatase activity. On the other hand, induction of gemmulogenesis by theophylline results in an increase (by 61%) in polyphosphate content of sponge tissue. An increase in polyphosphate content and a d…
Anti-HIV-1 activity of inorganic polyphosphates.
Human blood plasma, serum, peripheral blood mononuclear cells, and erythrocytes contain significant amounts of inorganic polyphosphates (ranging from 53 to 116 microM, in terms of phosphate residues). Here we demonstrate that at higher concentrations linear polyphosphates display cytoprotective and antiviral activity. Sodium tetrapolyphosphate and the longer polymers, with average chain lengths of 15, 34, and 91 phosphate residues, significantly inhibited human immunodeficiency virus type 1 (HIV-1) infection of cells in vitro at concentrations > or = 33.3 microg/ml (> or = 283-324 microM phosphate residues), whereas sodium tripolyphosphate was ineffective. In the tested concentration range,…
A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.
A method for determining inorganic polyphosphate, which is based on the Mn2+-induced quenching of the fluorescence of the calcium indicator fura-2, is described. The effect of Mn2+ ions on fura-2 fluorescence is gradually abolished in the presence of increasing concentrations of polyphosphate; this allows the quantification both of synthetic polyphosphates and of the naturally occurring polymer isolated from tissues or cells. The described method has some advantages compared to conventional procedures for detection of polyphosphates based on the metachromatic effect on toluidine blue. It can be applied for the determination of pyrophosphate, tripolyphosphate and other short-chain polyphosph…
Suppression of allograft rejection in the sponge Suberites domuncula by FK506 and expression of genes encoding FK506-binding proteins in allografts.
SUMMARY 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;…
Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis
Age-dependent studies show that the amount of inorganic polyphosphate in rat brain strongly increases after birth. Maximal levels were found in 12-months old animals. Thereafter, the concentration of total polyphosphate decreases to about 50%. This decrease in the concentration of total polyphosphate is due to a decrease in the amount of insoluble, long-chain polyphosphates. The amount of soluble, long-chain polyphosphates does not change significantly in the course of ageing. In rat embryos and newborns, mainly soluble polyphosphates could be detected. In rat liver, the age-dependent changes are less pronounced. The changes in polyphosphate level are accompanied by changes in exopolyphosph…
Inorganic Polyphosphate in Eukaryotes: Enzymes, Metabolism and Function
Inorganic polyphosphates (polyP) are linear polymers of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds. These polymers are widely distributed in nature, from archaebacteria, eubacteria, fungi, algae, and protozoa to higher plants and animals (for reviews, see Kulaev 1979; Wood and Clark 1988; Kornberg 1994, 1995). PolyP molecules are stable in neutral aqueous solutions, but are hydrolyzed by heat-treatment, and under acidic or alkaline conditions. The chain length of polyP may range from 3 to more than 1000 Pi residues; it can be analyzed on urea/polyacrylamide gels (Clark and Wood 1987; Lorenz et al. 1994a). In contrast to the linear polymer, branched inorganic p…
Purification and characterization of an exopolyphosphatase from Saccharomyces cerevisiae.
An exopolyphosphatase (polyphosphate phosphohydrolase; EC 3.6.1.11) activity that cleaves inorganic polyphosphates to orthophosphate has been purified to apparent homogeneity (> 95% pure) from Saccharomyces cerevisiae. The exopolyphosphatase is a monomeric protein with a polypeptide molecular mass of 28 kDa. The enzyme, which can be stabilized in the presence of Triton X-100, has a pH optimum of 7.5 and requires, for maximal activity, Co2+ or Mg2+ ions. In the absence of these ions, the exopolyphosphatase binds to polyphosphate but does not degrade it, allowing affinity purification of the enzyme on a polyphosphate-modified zirconia support. o-Vanadate, Cu2+, and Ca2+ are effective inhibito…
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-ch…
Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin
The major skeletal elements in the (Porifera) sponges, are spicules formed from inorganic material. The spicules in the Demospongiae class are composed of hydrated, amorphous silica. Recently an enzyme, silicatein, which polymerizes alkoxide substrates to silica was described from the sponge Tethya aurantia. In the present study the cDNA encoding silicatein was isolated from the sponge Suberites domuncula. The deduced polypeptide comprises 331 amino acids and has a calculated size of Mr 36 306. This cDNA was used as a probe to study the potential role of silicate on the expression of the silicatein gene. For these studies, primmorphs, a special form of aggregates composed of proliferating c…