Poly(gamma-glutamic acid)s are the major constituents of nematocysts in Hydra (Hydrozoa, Cnidaria).

The 2.80 +/- 0.20 mumol of anions found/mg of isolated and purified dry nematocysts (capsular secretory products of stinging cells) from Hydra make up the majority of the soluble capsular content. They are, in cooperation with corresponding cations, responsible for the generation and regulation of an internal osmotic pressure that amounts up to 150 bar (Weber, J. (1989) Eur. J. Biochem. 184, 465-476). The anions are organized as linear homopolymers of L-glutamic acids which are linked by gamma-carboxyl-alpha-amino amide bonds; the degree of polymerization is heterogeneous and dependent on the particular type of nematocyst. In situ the intracapsular glutamic acid monomer concentration is as high as 2 M. This is the first time that poly(gamma-glutamic acid)s, which are known to occur in some selected bacteria, are reported for eucaryotes. It is suggested that they may also be present as predominant components in nematocysts of other cnidarian species and thus might represent a class of compounds which is characteristic for a whole phylum of the animal kingdom.

Inotropic response elicited by nematocyst contents of Hydra oligactis (Coelenterata: Hydrozoa).

1. Intact, isolated nematocysts from the non-toxic, freshwater coelenterate Hydra oligactis contain soluble material(s) capable of producing a sustained increase in the rate of developed force in the vertebrate myocardium. 2. The positive inotropic effects of this material(s) appear grossly comparable to those described for Anthopleurin-A (AP-A) and Toxin II (ATX-II) from sea anemones. 3. The effects of the nematocyst material are distinct from those of known vasoactive peptides reported to occur in Hydra.

Hemolytic and toxic properties of Hydra attenuata nematocysts.

Crude extract prepared from isolated and purified nematocysts (stenoteles, desmonemes, isorhizas) of Hydra attenuata Pall. (Cnidaria, Hydrozoa) contains two main toxic proteins. The first is a hemolysin (100-200,000 mol.wt) which also causes initial spasmodic contractions in larval and adult specimens of Drosophila. Both, hemolytic and neurotoxic activities are inhibited by low concentrations of Triton X-100. The second protein (30-100,000 mol.wt), which is not susceptible to Triton causes long lasting paralysis leading to death of the test animals (LD50 approximately 5 mg crude nematocyst extract per kg). Neither of the toxins is identical with the previously described phospholipase.

Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra.

The presence of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the peduncle of Hydra was demonstrated by immunogold electron microscopy. Thin sections of Lowicryl-embedded tissue labeled with antisera to RFamide and 5-nm gold-conjugated, secondary antibody and of Epon-Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms/ml RFamide. These results are the first demonstration that the dense-cored vesicles of Hydra neurons contain a neuropeptide.

Computer aided evaluation of two-dimensional NMR spectra of proteins.

A computer program for the automatic evaluation of two-dimensional NMR spectra of peptides and proteins has been developed. The used strategy is described, the advantages and limits of this approach are discussed. The program was successfully tested on a COSY-spectrum of the neuropeptide Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe from hydra, resulting in a drastic reduction of the time needed for the evaluation of two-dimensional NMR data.

FMRFamide immunoreactivity is generally occurring in the nervous systems of coelenterates.

Abundant FMRFamide immunoreactivity has been found in the nervous systems of all hydrozoan, anthozoan, scyphozoan and ctenophoran species that were looked upon. This general and abundant occurrence shows that FMRFamide-like material must play a crucial role in the functioning of primitive nervous systems.

FMRFamide-like immunoreactivity in the nervous system of Hydra.

FMRFamide-like immunoreactivity has been localized in different parts of the hydra nervous system. Immunoreactivity occurs in nerve perikarya and processes in the ectoderm of the lower peduncle region near the basal disk, in the ectoderm of the hypostome and in the ectoderm of the tentacles. The immunoreactive nerve perikarya in the lower peduncle region form ganglion-like structures. Radioimmunoassays of extracts of hydra gave displacement curves parallel to standard FMRFamide and values of at least 8 pmol/gram wet weight of FMRFamide-like immunoreactivity. The immunoreactive material eluted from Sephadex G-50 in several components emerging shortly before or after position of authentic FMRFamide. The presence of FMRFamide-like material in coelenterates shows that this family of peptides is of great antiquity.

Bombesin-like immunoreactivity in the nervous system of hydra.

With immunocytochemical methods, nerve cells have been detected in Hydra attenuata containing bombesin-like immunoreactivity. These nerve cells are located in ectoderm of all body regions of the animal and are especially abundant in basal disk and tentacles. Radioimmunoassay of extracts of hydra demonstrated at least 0.2 pmol/g wet weight of bombesin-like immunoreactivity. The immunoreactive material elutes from Sephadex G-50 in a similar position to synthetic bombesin. The data show that bombesin-like peptides are among the phylogenetically oldest neuropeptides found so far.

Neurotensin-like immunoreactivity in the nervous system of hydra.

Neurotensin-like immunoreactivity is found in nerve fibers present in all body regions of hydra. The nerve fibers are especially numerous in the ectoderm at the bases of the tentacles and in the ectoderm at a site just above the foot. Radioimmunoassays of acetic-acid extracts of hydra, using various region-specific antisera towards mammalian neurotensin, show the presence of multiple neurotensin-related peptides. The amounts of these peptides vary between 1 and 350 pmol per gram wet weight. Gel filtration on Sephadex G-25 reveals a fraction of neurotensin-like peptides that crossreact equally well with an antiserum directed against sequence 1-8 and an antiserum directed against sequence 6-13 of neurotensin. This fraction elutes also at the position of neurotensin and might closely resemble the mammalian peptide. A fraction eluting with the void volume crossreacts preferentially with antisera directed against sequences 1-8 and 10-13 of neurotensin. Several components of apparent lower molecular weight than neurotensin crossreact preferentially with an antiserum against sequence 10-13. These last peptides represent the major portion of the neurotensin-like peptides in hydra.

Substance P-like immunoreactivity in the nervous system of hydra.

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.

Quantitative demonstration of cell surface involvement in a plant-animal symbiosis: lectin inhibition of reassociation.

The freshwater hydra, Hydra viridis is normally associated with Chlorella-like, algal symbionts which inhabit the host's digestive cells. Under experimental conditions bleached hydra will reassociate with algae harvested from green hydra, but not from our cultures of wild type Chlorella or strain NC64A which when originally isolated from Paramecium bursaria was symbiotically competent. Because of its demonstrated selectivity, the reassociation process is hypothesized to involve a recognition interface whose active participants are the algae cell wall and the digestive cell membrane. The data presented here confirm the hypothesis and suggest some potential molecular characteristics of the interacting partners. Concanavalin A (Con A), a plant lectin, used widely for similar studies in other systems totally inhibits reassociation; Wheat Germ Agglutinin (WGA), ricin and Lens culinaris lectin do so to a lesser degree. These results are consistent with the hypothesis that glycoproteins on the cells' peripheries are involved in cell-cell recognition in this system.

Human hypothalamus and intestine contain a hydra-neuropeptide.

From human hypothalamus and intestine a substance was isolated which has the same physico-chemical and biological properties as the head activator from hydra. The hypothalamus of an adult man contained activity corresponding to at least 10(8) hydra. An intestine of a 3-month-old human embryo contained approx. 10(5) hydra equivalents and that of a 6-month-old embryo 10(6). A gradual increase in content of head activator during development was also found in rat intestine.

Isolation of trimethyl amine oxide and other bases from the hydroid Tubularia larynx.

1. Trimethyl amine oxide, dimethyl amine and choline chloride have been isolated from the marine hydroid Tubularia larynx and identified using physical constants and spectral data. 2. The presence of two guanidine molecules has been demonstrated.

Isolation of a substance activating foot formation in hydra.

We have developed an assay for a substance from hydra that accelerates foot regeneration in the animal. This substance is specific for the foot as evidenced by the following findings: (1) It is present in the animal as a steep gradient descending from foot to head, paralleling the foot-forming potential of the tissue (2) It does not accelerate head regeneration, nor do the head factors of hydra discovered by Schaller (1973) and Berking (1977) accelerate foot regeneration. We propose that the foot-activating substance is a morphogen responsible for foot formation in hydra. The foot activator can be extracted from hydra tissue with methanol and separated from other known morphogens of hydra by gel filtration and ion-exchange chromatography. A substance with similar biological and physicochemical properties can be isolated from sea anemones.