The utilization of cnidarian nematocysts by aeolid nudibranchs: Nematocyst maintenance and release in spurilla

Some nudibranchs that feed on cnidarians are known to store nematocysts within cnidophage cells and use them for their own defense. Most of the nematocysts are in direct contact with the cytoplasm of the cnidophage. Nematocysts are not subjected to lysosomal enzymes because any phagocytic membrane that surrounded the nematocyst after engulfment does not persist. Cnidophage organelles are restricted to regions surrounding the nematocysts and may aid in the maintenance and development of the nematocysts. The release of cnidophages is initiated by a contraction of a dense muscle complex surrounding the cnidosac. Nematocysts do not discharge if the cnidophage membrane does not rupture upon release. A comparison of nematocyst maintenance in Spurilla neapolitana and nematocyst retention in other organisms is presented.     

Organelle survival in a foreign organism: Hydra nematocysts in the flatworm Microstomum lineare

Nematocysts are characteristic organelles of the phylum cnidaria. They are designated kleptocnidae when sequestered in animals that feed on cnidaria. Kleptocnidae are known for more than a century. Nevertheless it is still enigmatic how selected nematocyst types survive in the predator and how they reach their final destination in the foreign body. In the free-living Platyhelminth Microstomum lineare the fate of nematocysts of the prey Hydra oligactis was analyzed at the ultrastructural level and by fluorescence microscopy using hydra polyps that had been stained in vivo with the fluorescent dyes TROMI and TRITC. M. lineare digested hydra tissue in its intestine within 30?min and all nematocyst types were phagocytosed without adherent cytoplasm by intestinal cnidophagocytes. Desmoneme and isorhiza nematocysts were digested whereas cnidophagocytes containing the venom-loaded stenotele nematocysts started to migrate out of the intestinal epithelia through the parenchyma to the epidermis thereby traversing the subintestinal and subepidermal muscle layer. Within one to two days, M. lineare began to form a muscle layer basolateral around epidermal cnidophagocytes. Epidermal stenoteles survived in M. lineare for at least four weeks. The ability of epidermal stenotele nematocysts to discharge suggest that this hydra organelle preserved its physiological properties in the new host.     

Dynamic tuning of hair bundle mechanoreceptors in a sea anemone during predation

The sea anemone Haliplanella luciae (Cnidaria, Anthozoa) detects chemical and mechanical stimuli from prey. Hair bundle mechanoreceptors on the tentacles participate in regulating discharge of microbasic p-mastigophore nematocysts. Properly stimulated hair bundles sensitize the anemone to discharge nematocysts into objects that contact the tentacles. The hair bundle mechanoreceptors are composed of stereocilia derived from a multicellular complex. This complex consists of a single sensory neuron surrounded by two to four supporting cells. The mechanoreceptor is similar in many ways to vertebrate hair cells of the acousticolateralis system. However, anemone hair bundles are adjustable in structure and responsiveness according to the activity of two different chemoreceptors. One chemoreceptor binds N -acetylated sugars and the other binds amino compounds including proline. N -acetylated sugars induce lengthening of the hair bundle and a downward shift in frequencies that elicit maximal discharge of microbasic p-mastigophore nematocysts. Furthermore, N -acetylated sugars shift maximal discharge to smaller amplitude vibrations. Thus, N -acetylated sugars likely tune hair bundles so that small, swimming zooplankton stimulate maximal discharge. Proline leaks into the seawater from the hemolymph of wounded prey. Proline induces shortening of the hair bundle and shifts maximal discharge of nematocysts to higher frequencies and to larger amplitude vibrations. Thus, proline likely tunes hair bundles so that small, wounded, prey stimulate maximal discharge of nematocysts as they struggle to escape. Thus, suitably sized prey stimulate maximal discharge of microbasic p-mastigophore nematocysts upon first contacting the anemone tentacle and again upon attempting to escape.     

Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry

The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered.     

A sea anemone's environment affects discharge of its isolated nematocysts

Nematocysts were isolated from individuals of Calliactis tricolor maintained under different feeding schedules or in different salinities in an attempt to determine how these culture conditions influence the discharge of isolated nematocysts. In addition, the discharge frequencies of nematocysts isolated from two different populations of sea anemones found in two different environments were also compared. Undischarged acontial nematocysts were isolated by extrusion into 1 M sodium citrate and were then treated with 5 mM EGTA to initiate discharge. Nematocysts isolated from anemones maintained under three different feeding schedules showed significantly different responses to the test solution. Nematocysts isolated from anemones maintained in two different salinities did not differ significantly in discharge frequency. Nematocysts isolated from individuals from two separate populations of C. tricolor responded significantly differently to 5 mM EGTA and to deionized water, and these responses also depended upon the isolation solution used. Environmental conditions are known to have an impact on the physiological state of most organisms, but this is the first study providing evidence that the environment or feeding state of an anemone affects discharge of isolated nematocysts. Inherent differences in ionic and osmotic characteristics among nematocysts could explain some of the ambiguities when comparing past studies of isolated nematocyst discharge.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

Envenomation by the box-jellyfish Chironex fleckeri: how nematocysts discharge

The capsules of isolated mastigophores of C. fleckeri were impermeable to water and neutral red dye. After air drying, ca 30% discharged, most everting fully, when exposed to distilled water or sodium citrate but in a seawater medium only partial discharge was induced. The capsular contents of discharging mastigophores dyed strongly red with neutral red and showed metachromasy with toluidine blue. Electron micrographs revealed hexagonal arrays of granules ca 12 nm in diam. Evidence supports the view that a polymerization of the capsular material initiates tubule eversion and that complete eversion involves osmotic inflow of water and sustained compression of the capsular contents.     

Minicollagen-15, a novel minicollagen isolated from Hydra, forms tubule structures in nematocysts

Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated minicollagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C-terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diversification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule.     

Effects of controlled treatment with trypsin on the functional characteristics of isolated nematocysts of Calliactis parasitica and Aiptasia mutabilis (Cnidaria,Actiniaria)

Glycerol-isolated basitrichs of Calliactis parasitica responsive to thioglycolate had open apical flaps, while unresponsive capsules isolated with Triton X100 or by freezing had closed apical flaps. Limited treatment with trypsin induced the apical flaps to open without causing discharge, suggesting that nematocysts can maintain the resting condition even with open flaps. Trypsin-treated basitrichs acquire a high responsiveness to thioglycolate. Microbasic mastigophores of Aiptasia mutabilis are more responsive to distilled water after controlled trypsin treatment but the apical flaps are unchanged. Ca²⁺ is inhibitory regardless of trypsin treatment. It is proposed that the capsule tip may control the penetration of the discharging agents rather than providing mechanical resistance to inner pressure.     

Somatic embryogenesis and plant regeneration from litchi protoplasts isolated from embryogenic suspensions

High yields of protoplasts were isolated from litchi embryogenic suspensions, which were maintained by alternative culture in liquid and on solid media containing silver thiosulfate. Protoplasts in liquid culture and agarose beads were unable to divide sustainedly, whereas embedding of protoplasts in Ca-alginate supported cell division to microcalli and the direct formation of somatic embryos from protoplasts. Nurse cells of litchi further enhanced the culture efficiency when protoplasts were cultured in Ca-alginate beads. White non-hyperhydric somatic embryos were developed from protoplast-derived microcalli or proembryos, and 33.1% of white somatic embryos regenerated into plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

MsGC-II,a receptor guanylyl cyclase isolated from the CNS of Manduca sexta that is inhibited by calcium

We describe the cloning of a receptor guanylyl cyclase, MsGC-II, from the CNS of the insect Manduca sexta. Sequence comparisons with other receptor guanylyl cyclases show that MsGC-II is most similar to a predicted guanylyl cyclase in the Drosophila genome and to vertebrate retinal guanylyl cyclases. When expressed in COS-7 cells, MsGC-II exhibited a low level of basal activity that was nearly abolished in the presence of 10 micro m calcium. Incubation with either a mammalian guanylyl cyclase-activating protein or Drosophila frequenin resulted in only mild stimulation of activity, whereas incubation of COS-7 cells expressing MsGC-II with a variety of Manduca tissue extracts failed to stimulate enzyme activity above basal levels. Analysis of the tissue distribution of MsGC-II revealed that it is nervous system specific. In the adult, MsGC-II is present in neurons in the optic lobes, antennal lobes and cellular cortex, but it is most highly expressed in subsets of intrinsic mushroom body neurons. Thus, MsGC-II appears to be a neural-specific receptor guanylyl cyclase whose activity may be regulated either directly or indirectly by calcium.     

Impairment of ATP-linked reactions in mitochondria isolated from skeletal muscle of halothane-sensitive pigs

Oxygen consumption was depressed in mitochondria isolated from halothane sensitive pig (HP) muscle. The calculation of the respiratory control ratio (RCR) indicated that mitochondria were more affected at the site-I level of the respiratory chain. Calcium accumulation in these mitochondria was not altered when driven by the oxidation of succinate. This process was abolished when linked to ATP as a source of energy. ATP transport was completely inhibited in (HP) mitochondria.     

Lack of an effect of static magnetic field on calcium efflux from isolated chick brains

45Ca2+ efflux from neonatal isolated chick brains was measured. The brains were exposed to uniform or nonuniform static magnetic fields. The field intensity ranged from 200-900 mT. The exposure took place during incubation and/or when efflux was being measured. No difference appeared in the 45Ca2+ efflux between controls and exposed brains.     

Cation-induced aggregation of membrane vesicles isolated from vascular smooth muscle

Cations stimulated aortic muscle membrane aggregation with increasing potency according to their effective charge, e.g., K⁺2+3+, and the stimulation is reciprocally related to the apparent affinity for these cations. Divalent metal ion-induced membrane aggregation showed a dependence on the ionic radius, being optimal for Cd²⁺. Polyvalent cation-induced membrane aggregation was reversibly suppressed by high ionic strength as well as by metal ion chelators, irreversibly inhibited by the cross-linking agent glutaraldehyde, and enhanced by increasing concentrations of ethanol and increased temperature of the medium. When the pH is lowered below 6.0, membrane aggregation progressively increased with a concomitant decrease in cation-induced aggregation. The patterns of aggregation of microsomal membranes and further purified plasma membranes were almost identical whereas the aggregation of the heterogeneous mitochondrial membrane-enriched fraction was distinctly different in the initial rate of aggregation, its pH dependence, and metal ion concentration dependence. Our results indicate that cation-induced membrane aggregation can also be used to isolate a plasma membrane-enriched fraction from vascular smooth muscle.