Intracellular Ca2+ and Zn2+ levels regulate the alternative cell density-dependent secretion of S100B in human glioblastoma cells.

In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca(2+)- (Zn(2+)- and Cu(2+))-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca(2+) levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), the Ca(2+)-chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn(2+) levels. Treatment of cells with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn(2+)-binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca(2+) levels, and even more so, a decrease in Zn(2+) concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway.

Calcium-dependent translocation of S100A11 requires tubulin filaments.

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily.

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.

Supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas are characterized by a differential expression of S100 proteins.

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II-IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.

Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany.

A national interlaboratory study to validate two alternative methods to the Draize rabbit's eye test, co-ordinated by ZEBET at the German Federal Health Office (BGA), is described. The aim of the study is to classify chemicals according to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test. During the last two years 12 toxicology laboratories from industry, universities and other research institutions have tested 32 substances from a variety of chemical classes, characterized by a broad spectrum of locally irritating properties, using the NR/KB cytotoxicity test and the HET-CAM assay. Intra- and interlaboratory reproducibility of the two methods was investigated under standardized conditions. The so-far limited evaluation of the interlaboratory assessment phase of validation indicates that the results of the Draize rabbit's eye test correlate better with the results of the HET-CAM test than with those of the cytotoxicity test as far as false negative results are concerned. However, the intra- and interlaboratory reproducibility of the cytoxicity test is better than that of the HET-CAM test. The validation project has recently entered the stage of database development during which 150 chemicals will be tested in seven laboratories to provide information on whether and to what extent the NR/KB test and the HET-CAM test can replace the Draize rabbit's eye test for the classification and labelling of chemicals with regard to their eye irritation potential.

Studies on the design of animal tests for the corrosiveness of industrial chemicals.

Using a defined method for the determination of irritant and corrosive effects, the effects of varying the exposure time and the extent of occlusion were investigated and compared in rabbit skin experiments (by occlusive and semi-occlusive methods, each at exposure times of 1 hr and 4 hr). The results for 23 substances demonstrate that exposure for 1 hr normally leads to a realistic assessment of corrosiveness, in agreement with those given in the EEC 'Dangerous Substances Directive' (67/548/EEC; Off. J. Europ. Commun. 1967, 196, 1). With several substances, the 4-hr exposure leads to corrosive effects that do not occur under practical conditions. Moreover, a 4-hr exposure does not lead to a realistic hazard assessment in every case; some substances exhibit a corrosive effect in this test but are not classified as "corrosive" in the EEC Guideline Annex I, no. 1.1. Results using the semi-occlusive method did not usually differ from those obtained with the occlusive method. In general, the semi-occlusive method can be used, and in the case of volatile substances it is strongly advocated.

Febantel, a new broad-spectrum anthelminthic.

N-(2-[2,3-Bis-(methoxycarbonyl)-guanidino]-5-(phenylthio)-phenyl)-2-methoxyacetamide (febantel, Rintal) is a new compound which is highly active against various species of nematodes and cestodes in mice, rats, dogs, sheep and cattle. In sheep and cattle a single oral dose of 5 mg/kg resulted in an almost complete elimination of larval and adult intestinal nematodes as well as Dictyocaulus. The compound was well tolerated by all animals tested.

Pre-clinical toxicological studies with muzolimine.

Toxicological studies were carried out to assess the acute, sub-chronic and embryotoxicity of muzolimine, administered orally to a number of animal species. The results showed that muzolimine had only slight acute toxicity, of the same degree in the mouse, rat, rabbit and dog. In the sub-chronic toxicity studies over 90 days, the presenting symptom in rats and dogs was a pronounced diuresis and, apart from the kidney, no other organ or organ systems were impaired. Macroscopic and microscopic changes in the kidney were the pharmacological result of overdosage, i.e. excessive diuresis, and not due to typical toxic renal damage. Embryotoxicity studies on pregnant rats and rabbits showed that toxic (rat) or lethal doses (rabbits) had no embryotoxic or teratogenic effects.