Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

Didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate.

Extracts of samples of a Caribbean tunicate (ascidian, sea squirt) of the family Didemnidae inhibit in vitro at low concentrations the growth of DNA and RNA viruses as well as L1210 leukemic cells. The active compounds isolated from the tunicate, didemnins A, B, and C, are depsipeptides, and didemnin B (a derivative of didemnin A) is the component active at the lowest concentration in inhibiting viral replication in vitro and P388 leukemia in vivo.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties.

Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose.

Chordate muscle actins differ distinctly from invertebrate muscle actins. The evolution of the different vertebrate muscle actins.

A total of 30 actins from various chordate and invertebrate muscle sources were either characterized by full amino acid sequence data or typed by those partial sequences in the NH2-terminal tryptic peptide which are known to be specific markers for different actin isoforms. The results show that most, if not all, invertebrate muscle actins are homologous to each other and to the isoforms recognized as vertebrate cytoplasmic actins. In contrast the actin forms typically found in muscle cells of warm-blooded vertebrates are noticeably different from invertebrate muscle actins and seem to have appeared in evolution already with the origin of chordates. During subsequent vertebrate evolution there has been a high degree of sequence conservation similar or stronger than that seen in histone H4. Urochordates, Cephalochordates and probably also Agnathes express only one type of muscle actin. Two types, a striated muscle-specific form and a smooth muscle form, are already observed in Chondrichthyes and Osteichthyes. Later in evolution, with the origin of reptiles, both muscle actins seem to have duplicated again; the striated muscle type branched into a skeletal- and cardiac-specific form, while the smooth muscle form duplicated into a vascular- and stomach-specific type. These findings support the hypothesis that each of the four muscle actins of warm-blooded vertebrates are coded for by a small number and possibly only one functional gene.

New cyclic peptides with cytotoxic activity from the ascidian Lissoclinum patella.

The isolation and structures of a new patellamide (patellamide D) and two new lissoclinamides (lissoclinamides 4 and 5) from the aplousobranch ascidian Lissoclinum patella are described. Structures were determined largely by using two-dimensional NMR techniques and mass spectrometry. These peptides and other members of the patellamide and lissoclinamide families that have been reported previously are found within the obligate algal symbiont of the genus Prochloron. The cytotoxicities of the compounds toward fibroblast and tumor cell lines are reported. One of these compounds, lissoclinamide 4, is markedly more toxic than other members of the family. Structure-activity relationships are discussed.

Novel cytotoxic compounds from the ascidian Lissoclinum bistratum.

The isolation and structures of two new cyclic hexapeptides and two new macrocyclic ethers from the aplousobranch ascidian Lissoclinum bistratum are described. Their structures were determined by two-dimensional NMR techniques. The hexapeptides, named bistratamide A and bistratamide B, differ only by the presence or absence of one double bond. They were tested for cytotoxicity toward human fibroblast and tumor cell lines and displayed similar toxicities to the octapeptides called patellamides from Lissoclinum patella. The peptides are found within the obligate algal symbiont Prochloron but clearly differ from peptides isolated from the same Prochloron of L. patella. The macrocyclic ethers isolated from L. bistratum are exceedingly potent in cytotoxicity. They have been named bistratenes A and B, and structures for these compounds are proposed.

Sugar specific cellular lectins of Phallusia mamillata hemocytes: purification, characterization and evidence for cell surface localization.

Cellular lectins (CLs) of Phallusia mamillata were demonstrated in protein preparations obtained by salt fractionation from hemocytes sonicated in a suitable medium. Since the lectins from the precipitated fraction bind sugars containing D-galactosyl groups, they were purified by affinity chromatography on Sepharose. SDS-PAGE under reducing conditions showed that CLs are formed of two components of apparent MWs approximately 36,900 and 35,090 and thus differ from serum lectins (SLs) (MW about 62,200). The "shrinkage" observed when SLs were examined under nonreducing conditions suggest the presence of intrachain disulphide bonds which can affect the molecular structure of the SLs. CL-SL differences were also revealed by the nonidentity reaction of the immuno-precipitate in immunodiffusion using an anti-SL immune serum. The capacity of hemocytes to form rosettes or clumps with erythrocytes demonstrated that they possess alpha-lactose specific CLs on their surfaces.

Immunoreactive human calcitonin-like molecule in the nervous systems of protochordates and a cyclostome, Myxine.

A molecule very closely resembling human calcitonin immunologically and chromatographically was extracted from the nervous systems of several protochordates and a cyclostome, Myxine. The presence of human calcitonin-like molecules in the nervous systems of primitive chordates suggests that they have some function in the nervous system of these species and that the bone-regulating function of the calcitonins may have arisen much later in the vertebrates.

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization.

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.

Removal of troponin C and desensitization of myosin B from ascidian smooth muscle by treatment with ethylene diamine tetraacetate.

On treatment with 10 mM EDTA at 30 degrees C, protein of 18,000 daltons was released from myofibrils, thin filaments and myosin B prepared from the smooth muscle of an ascidian, Halocynthia roretzi. This protein was purified from the EDTA extract of myofibrils by differential centrifugation, freeze-drying and gel-filtration. Based on its molecular weight, electrophoretic mobilities in the presence and absence of Ca2+ and other properties, it was identified as troponin C. By EDTA treatment, ascidian myosin B lost the Ca2+-sensitivity of Mg2+-ATPase, and EDTA-treated myosin B recovered the sensitivity by mixing with the EDTA extract of myosin B in the presence of Mg2+. Gel-electrophoretic patterns indicated that desensitization and resensitization of ascidian myosin B were accompanied by the removal and binding of troponin C. These results indicate that ascidian smooth muscle is regulated by a troponin-tropomyosin system, and desensitization induced by EDTA treatment is due to the removal of troponin C but not the release of the light chains of the myosin molecule. Based on these findings, we have established a simple method for the purification of troponin C from ascidian smooth muscle.

Troponin and its components from ascidian smooth muscle.

Troponin was isolated from the thin filaments of ascidian smooth muscle and separated into three components by ion-exchange chromatography, the molecular weights of which were 33,000, 24,000, and 18,000, respectively. The three components were designated as troponin t (TN-T), troponin I (TN-I), and troponin C (TN-C) in order of molecular weight, since each component had properties similar to those of the respective components of vertebrate skeletal-muscle troponin. The ascidian troponin or the mixture of the three components conferred Ca2+-sensitivity on reconstituted rabbit actomyosin in the presence of tropomyosin. One of the characteristics of the ascidian troponin was Ca2+-dependent activation of actin-myosin interaction in collaboration with tropomyosin, whereas its inhibitory action on the actomyosin ATPase in the absence of Ca2+ was less remarkable. From this, it is concluded that in the ascidian smooth muscle actin-myosin interaction is regulated by an actin-linked troponin-tropomyosin system, but the ascidian troponin acts as a Ca2+-dependent activator of an actomyosin system.

Cholecystokinin (CCK)/gastrin-like immunoreactive neurones in the cerebral ganglion of the protochordate ascidians Styela clava and Ascidiella aspersa.

Antisera raised against the COOH-terminal sequence of mammalian CCK/gastrin were used to ascertain the distribution of CCK/gastrin-like immunoreactive cells in the cerebral ganglion of two ascidian protochordates. Styela clava and Ascidiella aspersa. the cell bodies were found to have a specific regional distribution in Ascidiella, but not in Styela. In addition to central immunoreactive nerve fibres, a number of peripherally located immunoreactive fibres was found. These observations support the idea that some centrally originating protochordate neuronal peptides may have a peripheral role and give weight to the hypothesis that many vertebrate brain-gut peptides had their origin in the neuronal elements of more primitive species.

Sulphated CCK-8-like peptides in the neural ganglion of the protochordate Ciona intestinalis.

Immunochemical studies were carried out on extracts of the neural ganglion from the ascidian Ciona intestinalis in order to the characterize the peptide(s), which react with antibodies against the C-terminal sequence common for the mammalian hormones, cholecystokinin (CCK) and gastrin. Radioimmunoassays specific for the sulphotyrosyl-containing N-terminus of CCK-8, for the common alpha-carboxyamidated C-terminus and for gastrin were used to monitor gel chromatography and reverse-phase HPLC of the extracts. Only neutral extracts contained immunoreactive material (634 (524-785) pmol eqv.CCK-8/g) (mean and range, n = 4)). HPLC revealed a small peak eluting almost like CCK-8 and a larger peak eluting earlier. By subsequent gel chromatography the larger peak eluted in the same position as sulphated CCK-8. The material was recognized almost equally by the N- and C-terminal CCK radioimmunoassays, whereas the specific C-terminal gastrin radioimmunoassay did not measure the peptides. Treatment with arylsulphatase removed the binding to the antiserum specific for the sulphotyrosyl-containing sequence of CCK. The results indicate that the ganglion of Ciona intestinalis contains a tyrosyl-sulphated peptide resembling mammalian CCK-8.

Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis.

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.

Structure of a unique sulfated alpha-L-galactofucan from the tunicate Clavelina.

A purified sulfated alpha-L-galactofucan from Clavelina sp. has been studied before and after desulfation, using periodate oxidation, methylation analysis, and n.m.r. spectroscopy, and shown to be composed mainly of 3-sulfated 4-linked alpha-L-galactopyranosyl residues. There was also a small proportion of 3-sulfated 4-linked alpha-L-fucose residues.

Structural studies of a sulfated L-galactan from Styela plicata (Tunicate): analysis of the Smith-degraded polysaccharide.

The tunic of the ascidian Styela plicata is rich in a high molecular weight sulfated-L-galactan called the F-1 fraction. This polysaccharide is of complex structure and is highly branched. In this study we undertook detailed structural analysis of the F-1 fraction that was submitted to the Smith degradation procedure which, for this polysaccharide, results mainly in the elimination of the branches. By methylation, and one- and two-dimensional n.m.r. analysis of the Smith-degraded and desulfated Smith-degraded F-1 fraction, we determined the main structure of this polymer. It is composed of a core of alpha-L-galactopyranose units, sulfated at C-3 and glycosidically linked through position 1----4. The nonsulfated, nonreducing end units are branched at C-2 of the sulfated L-galactoses.

[Anti-tumor natural products isolated from marine organisms].

Marine organisms comprise over half a million species. Due to their unusual living environment as compared with terrestrial organisms, marine organisms produce a variety of metabolic substances which often have various unprecedented chemical structures. In recent years, an increasing number of marine natural products have been reported to exhibit various biological activities such as antimicrobial, physiological, and pharmacological ones. Some metabolites have also been noted by their significant toxicities. The effort to find out anti-tumor substances from marine organisms has been also undertaken in recent years, and several novel compounds (e.g. peptide, polyether, alkaloid, prostanoid etc.), with anti-tumor activities, have been isolated from marine sponges, octocorals, marine algae, tunicates, nudibranchs, bryozoans, and so on. In this article, those chemically clarified anti-tumor compounds isolated from marine organisms were reviewed.

[A comparative study of the biogenic amine receptors in the muscle tissue of mollusks, echinoderms and tunicates]. / Sravnitel'noe izuchenie retseptorov biogennykh aminov v myshechnoi tkani molliuskov, iglokozhikh i obolochnikov.

Smooth muscles of Mollusca, Echinodermata and Tunicata contain one class of adrenoreceptors with the dissociation constant and maximal specific binding 2.5 and 82 in Anodonta, 2.3 and 320 in Holothuria, 4.9 pM and 232.5 fmol/mg of protein in ascidia. Catecholamines and their antagonists can be ranged in the row as follows: isoproterenol greater than adrenalin greater than propranolol-noradrenaline greater than phentolamine. Negative regulation of the beta-adrenoreceptor affinity to isoproterenol by means of guanine nucleotides (GN) was shown. The muscular tissues of Mollusca, Echinodermata and Tunicata have only one class of the serotonin receptors with the dissociation constant and maximal specific binding 120 and 13.2 in Anodonta, 88 and 192 in Holothuria, 2.6 pM and 54 fmol/mg of protein in ascidia. The GTP negative regulation of serotonin receptors affinity to the hormone was found. The GN regulation of the above receptors affinity to agonist suggests that muscle tissue of the above species has specific GTP [correction of GTR]-binding proteins capable of coupling with these receptors.