Are echinoderms of interest to biotechnology?

The huge potential of echinoderms as a so far fairly untapped source of bioactive molecules is described. Examples are presented that show the usefulness of echinoderm-derived molecules for therapeutic application in selected fields of cancer research, in the control of bacterial growth as substances with new antibiotic properties, and finally in the context of technical applications such as antifouling substances. The molecules described here are but the mere beginning of a commercial exploitation of echinoderms and may incite a deeper involvement of biotechnology-oriented research in this material.

Participation of the Ca(2+)-calmodulin-activated Kinase II in the control of metaphase-anaphase transition in human cells.

Treatment of HeLa S3 cells growing in suspension, and of endothelial cells ECV 304 growing as a monolayer, with the inhibitor of the Ca(2+)-calmodulin activated Kinase II KN-93, blocks cells at metaphase for 15 min (HeLa cells) and 30 min (ECV cells). Thereafter cells resume mitosis and enter anaphase. The inactive isomer KN-92 does not show such effects. The results show the involvement of the CaM K II system in the regulation of the metaphase-anaphase transition whereby the activation of the Kinase II, in particular by calmodulin appears to be affected, the residual autophosphorylation of the CaM K II system apparently sufficing after 15 to 30 min to release the cells into anaphase. The results are compared with the metaphase-blocking effects of the noble gas xenon, where the xenon-induced block can be overcome by small intracellular increases of Ca(2+), thus indicating the CaM K II system as a possible target for xenon.

Xenon induces metaphase arrest in rat astrocytes.

The use of xenon as an almost ideal anesthetic with very little side effects is gaining clinical acceptance, yet its effects on the cellular level are still unclear. It affects intracellular Ca2+-homeostasis but up to now no cellular event or Ca2+-signaling system has been described to be specifically sensitive to xenon. Here we report for the first time a specific effect of xenon on astroglial cells not found with another volatile anesthetic, isoflurane, nor with helium nor with N2: treatment of primary astroglial cells from embryonic rat brain with xenon induces, apart from a slight retardation of the cell cycle, a block at metaphase. Upon removal of xenon cells arrested at metaphase complete their mitosis normally. Even under continuous exposure to xenon, cells can be rescued from metaphase arrest by a small and transient increase in intracellular Ca2+; cells enter anaphase despite the presence of xenon and complete cell division, exhibiting normal rate of chromosome movement and normal chromosome separation. These results suggest that xenon interferes with some Ca2+-dependent regulatory system required for the metaphase-anaphase transition; taking into account its anesthetic effects, xenon may be also involved in the control of glia-mediated signaling transfer.

Reduction of cell density dependent antigenic properties of intracellular Ca(2+)-regulating membranes by isoflurane in human endothelial cells.

An established cell line of human umbilical vein endothelial cells displays a pronounced shift in the distribution of intracellular Ca(2+)-regulating membranes as the cells grow towards confluence. In sparsely populated cultures a linearly oriented punctate pattern of vesicle-like structures is observed similar to the distribution found in many other eucaryotic cells. As the cell population increases, the membranes condense around the nucleus. In completely confluent cultures when the cells cease to proliferate, the antigen is no longer detectable by immunofluorescence; its absence is confirmed by Western blotting experiments. Double-labeling with antitubulin or phalloidin shows that the distribution of microtubules is not related to the distribution of the Ca(2+)-regulating membranes whereas the actin fibers are superimposable onto the linearly oriented punctate vesicle-like structures. If proliferating cells are treated with the volatile anesthetic isoflurane, the Ca(2+)-regulating membranes can no longer be detected with the antibody; however, in Western blots the antigen is still present. Staining is restored after the removal of isoflurane. Such a temporary disappearance of the immunocytochemical staining might be explained by a transient oxidation and subsequent configuration change of the antigen, rendering the epitope inaccessible to the antibody, since a treatment with low concentrations of NaBH4 of cells exposed to isoflurane restores the access of the antigen to the antibody.

Xenon-induced inhibition of Ca2+-regulated transitions in the cell cycle of human endothelial cells.

Xenon is an anesthetic with very few side-effects, yet its targets at the cellular level are still unclear. It interferes with many aspects of intracellular Ca2+ homeostasis, but so far no specific event or defined regulatory complex of the Ca2+-signaling system has been identified. Specific effects of xenon were found by investigating its effects on the cell cycle in human endothelial cells: there is a relationship between two cell cycle transition points, their regulation by Ca2+, and specific blocks induced by xenon. Within the group of substances studied (xenon, isoflurane, desflurane, helium, and N2), only xenon blocks the cells almost completely at the G2-M transition after a 2-h treatment; those cells that slip through this block are then arrested at metaphase. If xenon is removed, cells that have been accumulating at the G2-M boundary move into mitosis, and cells blocked at metaphase complete their mitosis normally. No such specific block of the cell cycle was found with the other substances studied. An artificial increase of intracellular Ca2+ in the submicromolar range, using a very low dose of the Ca2+ ionophore ionomycin, or a threefold increase of the external Ca2+ concentration suffices to lift the xenon-induced metaphase block; the cells enter anaphase despite the presence of xenon and complete cell division. Thus, the specific but completely reversible inhibition by xenon of the G2-M transition and the block at metaphase suggest an interaction with a Ca2+-dependent event involved in the control of these processes. The results are consistent with the hypothesis that suppression of Ca2+ signals can be considered as a common denominator of the effects of xenon on the cell cycle and on the neuronal system during anesthesia.

Human serum pyroglutamyl-beta-naphthylamide hydrolyzing activity during development and aging.

Pyroglutamyl-beta-naphthylamide hydrolyzing activity (pGluHA) is reported to be capable of removing the amino-terminal pyroglutamic acid residue from peptides (e.g. TRH or GnRH) and artificial substrates. However, its functional role in serum is not yet understood. The aim of the present study was to analyze the activity of pGluHA in human serum during development and aging, in an apparently healthy population of 139 men and 148 women. To measure pGluHA we used pGlu-beta-naphthylamide as the substrate. Sex differences and age-related changes were observed in men and women. In addition, the developmental profile was notably different between men and women. In men, activity increased steadily until full sexual maturity, but did not change substantially after puberty. In women, activity increased significantly in advanced ages but there were no significant changes in the rest of the age groups tested. Significant sex differences were observed in subjects 46-65 years old, the activity being higher in men than women. In the total population, a significant direct correlation was observed between pGluHA and age. Considered independently, men and women also showed a highly significant direct correlation between pGluHA and age. These results may reflect changes in the functional status of its circulating substrates during development and aging.

The centrosomal protein centrosomin A and the nuclear protein centrosomin B derive from one gene by post-transcriptional processes involving RNA editing.

The identification of a gene encoding concomitantly a nuclear protein and an intrinsic centrosomal protein further emphasizes the close and presumably developmental relationship between the cell nucleus and the centrosome. Screening of a murine RNA-based cDNA library with an antiserum to a centrosomal protein and rescreening with the insert of an initial clone released two complete cDNAs (1.2 kbp and 2.2 kpb) coding for proteins with notable characteristics. The amino-terminal sections of centrosomin A (276 amino acid residues, molecular mass 34.5 kDa) and of centrosomin B (447 amino acid residues, molecular mass 54.8 kDa) are identical over 272 amino acid residues. The carboxy-terminal section of the larger protein comprises additional 175 amino acid residues including nuclear location signals. The mRNAs encoding centrosomin A and B derive from a single gene. Chromogenomic DNA as template and primer pairs complementary to the sequence which is identical in centrosomin A and B cDNAs results in amplification of only one DNA fragment. Moreover, one exon of the genomic sequence and the centrosomin B-encoding cDNA sequence include a G which is deleted in the centrosomin A-encoding cDNA. Accordingly, the two mRNAs are the products of either alternative splicing or alternative polyadenylation in combination with RNA editing. The recombinantly expressed chimeric protein consisting of centrosomin A and the green fluorescent protein from Aequorea victoria accumulates in centrosomes while the corresponding fusion protein with the centrosomin B sequence is transported into nuclei.

Microtubule tracks can be detected in mouse oocytes with an antibody directed against a calcium transporter.

In metaphase II-arrested mouse oocytes, most microtubules are found in the meiotic spindle, a structure that remains stable for hours despite microtubule instability. Microtubule organizing centres (MTOCs) are present at the poles of the spindle and in the cytoplasm, but the latter nucleate very few microtubules. This particular organization of the microtubule network enabled us to observe the unexpected behaviour of a protein that can associate with microtubules. We compared the distribution of a mitosis-activated calcium transport system with that of the microtubule network, by immunofluorescence, using two monoclonal antibodies, one directed against a component of the calcium transport system (7/13), and the other against the common tyrosinated form of alpha-tubulin (YL1/2). The 7/13 staining was associated with the spindle microtubules and with the kinetochore area. In addition, we observed many asters in the cytoplasm, around the cytoplasmic MTOCs. The majority of these asters were not stained with the antitubulin antibody. Moreover, these 7/13 asters either disappeared after nocodazole treatment or were enlarged after taxol treatment. Using a confocal microscope, we observed single fibres that were stained with both antibodies: the extremity furthest from the MTOC (corresponding to the + end of the microtubule) being detected by the 7/13 antibody only. All these observations suggest that the 7/13 antigen is associated with microtubule tracks that persist a few minutes after microtubule depolymerization. The possible role of these tracks in microtubule regrowth is discussed.

cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/M phase-prevalent protein.

Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (lambda CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 x 10(3) M(r)) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.

Murine cDNAs coding for the centrosomal antigen centrosomin A.

Screening of an induced Ehrlich ascites cell-derived lambda gt11 cDNA library with an antibody (GP1), immunoreacting specifically with centrosomal antigen(s) of interphase and mitotic cells of different species, released a partial cDNA clone (lambda P10A) encoding the carboxy-terminal section of a centrosome-specific antigen. This specificity of the clone lambda P10A could be verified by lacZ-directed antigen expression from Escherichia coli Y1089 lysogenized with the recombinant phage lambda P10A and subsequent production of centrosome-specific antibodies by means of the recombinant antigen. Using the lambda P10A insert as a probe, two types of cDNA clones were identified in a lambda gt10 cDNA library by plaque-hybridization. The inserts of PN1 type clones were 1.2 kb (kilobases) and those of PN5 type clones were 2.2 kb in length. The DNA sequence of a PN1 type clone revealed its full-length cDNA nature. The open reading frame of PN1 encodes a rather hydrophilic and highly charged 34.5 x 10(3) Mr polypeptide comprising short but apparently significant strings of 100% sequence identity with the major nuclear lamina polypeptides lamins A/C and lamin B. Restriction enzyme mapping of PN1 and PN5 inserts, cross-hybridization experiments and comparison of overlapping DNA sequences indicate that the 1.2 kb and 2.2 kb cDNAs code for the same 34.5 x 10(3) Mr polypeptide, termed centrosomin A. Western blots of Ehrlich ascites cell proteins show a second, larger GP1 antigen (centrosomin B) whose cDNA has not been cloned. It remains to be investigated whether centrosomin B is encoded by a second mRNA or whether it reflects an oligomeric or a postranslationally modified form of centrosomin A.

Rhythmic oscillations of cytosolic free calcium in rat C-cells.

The relationship between stimulation of single C-cells (rMTC-6-23 cell line) with extracellular calcium, glucagon or 8-bromo-cAMP and fluctuations of intracellular free calcium concentration was studied. After pretreatment of rMTC cells with either 1 microM glucagon (30-60 min) or 1 mM 8-bromo-cAMP (5 min) [Ca2+]i started to oscillate when extracellular calcium was raised to 3 mM. These fluctuations in [Ca2+]i could be stopped by chelating the external calcium with EGTA or by adding calcium channel blockers. The voltage-dependent calcium channels in the plasma membrane seem to play a major role in maintaining the oscillations of [Ca2+]i.

Microtubules and Ca2+-sequestering membranes in the mitotic apparatus, isolated by a new method.

The mitotic apparatus of sea urchin embryos was isolated using a polyethylene glycol (PEG)/EGTA-medium. Such a procedure preserves the birefringence and the Ca2+ lability of the isolated mitotic apparatus. The method of isolation gives good preservation of the microtubules and of the intracellular Ca2+-transport system as visualized by a monoclonal antibody to a 46-kDa protein. Triple fluorescence studies allow a comparison of the relative locations of microtubules, Ca2+-sequestering membranes and chromatin (by Hoechst 33342) in the mitotic apparatus. We find that the Ca2+-sequestering membranes are concentrated mainly in the centers of the asters and do not follow the distribution of microtubules in the mitotic apparatus. Regulation of microtubules by Ca2+ may not depend on immediate contiguity of microtubules and the Ca2+-regulating sites.

Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2.

A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.

Inhibition of mitosis by an antibody to the mitotic calcium transport system.

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice.

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.

Visualization of the Ca-transport system of the mitotic apparatus of sea urchin eggs with a monoclonal antibody.

Monoclonal antibodies have been obtained to components of Ca(2+)-sequestering vesicles from the endoplasmic reticulum of HeLa cells by isolating hybridomas that were generated by the in vitro immunization of lymphocytes followed by fusion with plasmocytoma cells. One of these monoclonal antibodies specifically labels punctate structures which appear in the mitotic apparatus of sea urchin eggs at the beginning of prophase and disappear upon the completion of cytokinesis. The antibody inhibits the Ca(2+) uptake of the membrane system in vitro. It reacts with one 46-kDa protein out of the complex protein mixture from the membrane fraction. We take all this as evidence that in fact a specific Ca(2+)-transport system is part of the mitotic apparatus, that such a system is very conserved, and that it is most probably derived from the endoplasmic reticulum.

The so-called anticalmodulins fluphenazine, calmidazolium, and compound 48/80 inhibit the Ca2+- transport system of the endoplasmic reticulum.

We present evidence that a Ca2+- transport system of the endoplasmic reticulum with the "mitotic" Ca2+- ATPase as an essential component is another target for the anticalmodulin drugs fluphenazine, calmidazolium, and compound 48/80. Furthermore we show by affinity chromatography that there is a direct interaction between the solubilized Ca2+- ATPase and fluphenazine. Since the Ca2+- uptake system as well as the solubilized Ca2+- ATPase are calmodulin- free, the effect of fluphenazine, calmidazolium and compound 48/80 may be understood as a result of the interaction between these drugs and the Ca2+- ATPase. We propose that there are calmodulin- like sequences in the molecule of the Ca2+- ATPase. The inhibitory effect of these three drugs can be then explained by their recognition of the calmodulin- like structures.

Cell cycle specific variations in transport capacity of an isolated Ca2+-transport system.

From sea urchin eggs as well as from mammalian cells a Ca2+-transporting system is described in its properties. One of its main components is the "mitotic" Ca2+-ATPase. If its activity is studied during the cell cycle of fertilized sea urchin eggs, fluctuations of the Ca2+-uptake capacity are found with a maximum in every cell cycle at mitosis. Additionally, only in the first cycle after fertilization, another activity increase occurs at the time of spermaster formation. This system, then, seems to qualify for one of the main regulators of the mitotic process.

Localization of an intracellular membrane-bound Ca2+-ATPase in PtK-cells using immunofluorescence techniques.

Monospecific antibodies to an intracellular membrane-bound Ca2+-ATPase were used to localize the enzyme in PtK-cells in interphase and in mitosis as well. In interphase the protein is distributed as small dots and rods in the cytoplasm with an increased concentration around the nucleus. Neither the plasma membrane nor the nuclear envelope are stained. In mitotic cells the Ca2+-ATPase is localized around the spindle rather than in it. The results are in agreement with the proposed function of enzyme as an essential part of the intracellular Ca2+-regulating system controlling Ca2+ in the respective domains of the cell.