Integrin and talin in the jellyfish Podocoryne carnea

We have isolated an integrin-beta and -alpha subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life-cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT-PCR demonstrates that all three genes are maternal messages and continuously expressed in the life-cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co-expression of both integrin subunits in the different life-stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up- or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.     

Effects of transketolase cofactors on its conformation and stability

In studying transketolase (TK) from Saccharomyces cerevisiae, the majority of researchers use as cofactors Mg(2+) and thiamine diphosphate (ThDP) (by analogy with other ThDP-dependent enzymes), whereas the active site of native holoTK is known to contain only Ca(2+). Experiments in which Mg(2+) was substituted for Ca(2+) demonstrated that the kinetic properties of TK varied with the bivalent cation cofactor. This led to the assumption that TK species obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, adopt different conformations. Kinetic study of the H103A mutant yeast transketolase. FEBS Letters 567, 270-274]. Analysis of far-UV circular dichroism (CD) spectra and of data, obtained using methods of thermal denaturing, differential scanning calorimetry (DSC) and tryptophan fluorescence spectroscopy, corroborated this assumption. Indeed, the ratios of secondary structure elements in the molecule of apoTK, recorded in the presence of Ca(2+) or Mg(2+), respectively, turned out to be different. The two forms of the holoenzyme, obtained by reconstitution from apoTK and ThDP in the presence of Ca(2+) or Mg(2+), respectively, also differed in stability: the holoenzyme was more stable in the presence of Ca(2+) than Mg(2+).     

The homeobox gene Otx of the jellyfish Podocoryne carnea: role of a head gene in striated muscle and evolution

In many bilaterian animals members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain and Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. In contrast, functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later.     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.     

Formation and discharge of nematocysts is controlled by a proton gradient across the cyst membrane

Cnidaria catch and kill their prey by means of nematocysts. A nematocyst consists of a capsule containing a coiled tubule. On triggering, the cyst extrudes this tubule in an extremely rapid manner. The mechanisms and driving forces of discharge are still unknown. We found nematocysts of various cnidarians to be acidic inside and propose that the pH difference between cyst matrix and cytoplasm drives the discharge of cysts. For large cysts of Aiptasia we calculated that the internal concentration of protons and protonated carboxyl groups is about 5 M. Cysts contain polyacids in a high concentration. At a low pH several of the carboxyl groups of the polymer are uncharged. The carboxyl groups dissociate when, on triggering, the proton concentration becomes balanced across the cyst membrane. The speed of protons in water is extremely rapid. Thus, the equilibration of the proton concentration initially results in a negative net charge within the cyst and therefore in a sudden electrostatic repulsion between the dissociated carboxyl groups of the polymer. This causes an increase in the pressure of the matrix against the cyst wall. We suggest that this nonosmotic pressure increase causes the first and extremely rapid step of discharge. We propose that in a second step cations and water are taken up, generating an increase in osmotic pressure. A change in the pH value may also facilitate the invagination and evagination, respectively, of the tubule.     

Light and electron microscopic localization of a monoclonal antibody in neurons in situ in the head region of Hydra

A mouse monoclonal antibody to Hydra attenuata was used to demonstrate immunoreactive product in neurons in situ, in both whole mount and sectioned hypostomes and tentacles of H. oligactis and H. littoralis. Immunoreactive cells were concentrated around the mouth and scattered along the length of the tentacles. In the hypostome, nerve cells sent one or more processes orally and the others aborally but the processes were more distinctly stained in H. oligactis. A thin strand of five to six perihypostomal neurons was present close to the hypostome-tentacle junction. In the tentacles, neurons with long processes contacted up to five different batteries of nematocysts. Neural processes were associated with nematocyst batteries in three ways: 1) forming a perikaryal loop to encircle a centrally located stenotele, 2) branching at a distance from the perikaryon to contact a variety of nematocysts, and 3) terminal branching by one or more neurons with contacts on one to several nematocysts within a battery. Immunocytochemical localization of neurons in Hydra by light microscopy was correlated for the first time with electron microscopy. Peroxidase-antiperoxidase (PAP)-positive sensory cells were concentrated around the mouth opening. PAP-positive ganglion cells were predominant in the tentacles. Sensory cells were elongate or spindle-shaped (unipolar), triangular with two oppositely directed processes (bipolar), and multipolar (tripolar or tetrapolar) with one of the processes extending to the epidermal surface. Ganglion cells were either unipolar or bipolar or multipolar, with neurites paralleling the mesoglea and occasionally having processes abut on it.     

Cytological studies of the nematocysts of Hydra. I. Desmonemes,isorhizas, cnidocils,and supporting structures

Entire hydras or tentacles were fixed in OsO(4) or in KMnO(4) and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

The fibrous system of the extracellular matrix of Podocoryne carnea and its degradation by the subumbrellar plate endoderm demonstrated by a monoclonal antibody

Homogenized fragments of crude umbrellar material of the hydromedusa Podocoryne carnea was injected into BALB/c mice. The immunization resulted in the isolation of a monoclonal antibody designated 3A1 which specifically binds to fibrils in the mesogloeal extracellular matrix (ECM) of hydromedusae. In vivo, the architecture and the ultrastructure of the fibrous system in the outer mesogloea (outer ECM) of Podocoryne carnea, and its degradation under in vitro conditions have been described by morphological and immunological criteria. In vivo, 120-150 A thick, striated fibrils (with periodicities up to 50 nm) form a threedimensional network which fills in the entire outer ECM. Vertical fibers (up to 150 nm in diameter) penetrate the three-dimensional network and branch at the subumbrellar and the exumbrellar side. The vertical fibers show uniform distribution over the entire outer ECM. The branches impinge on a dense matrix (about 30 nm in thickness) covering the exumbrellar and subumbrellar surface. In vitro, the fibrillar system does not alter in its basic pattern, neither in isolated outer ECM, nor in portions of outer ECM which is either covered by the exumbrella, or which is attached to both: the exumbrella at the outside, and the subumbrellar plate endoderm at the inner side. After removal of the exumbrellar cells in the latter portions, a characteristic pattern of selective degradation of the outer ECM by the endodermal cells is observed. This process involves three distinct steps: an initial extracellular condensation within the ECM fibrillar network, followed by intercellular internalization of the fibrillar elements and subsequent endocytosis of ECM material. The first step immediately follows the removeal procedure of the exumbrellar cells and is completed within minutes. This process cannot be interrupted by dihydrocytochalasin B (H(2)CB). The second step lasts 24-48 hr, is mediated by cell mechanisms, and can be stopped by H(2)CB. The third step is a slow process (of up to 14 days). It involves intercellular degradation of fibrillar material, endocytosis, and completion of digestion within lysosomes.     

Inorganic Nutrient Concentrations and Chlorophyll in the Euphotic Zone of Lake Tanganyika

The taxonomic value of nematocyst size in sea anemones is still being assessed. We evaluate size distribution of nematocysts of one type in a single individual anemone. Length of unfired nematocysts was measured along the column, tentacles, and actinopharynx of a preserved specimen of Actinodendron arboreum (Quoy & Gaimard, 1833). Mean, range, minimum, and maximum length of nematocysts vary along the column, those in the middle region being least variable. The length of nematocysts in mature (split) acrospheres is less variable than in immature (unsplit) acrospheres. There is significant variability between nematocysts in tentacles of the primary and quaternary cycles, and along a tentacle, the middle being least variable. Size distribution of actinopharynx nematocysts is complex. The results of this study suggest that assembling data on nematocysts from multiple individuals for taxonomic purposes should be used with an awareness that sampling site can be an important variable. Ideally, the position of tissue sampled should be documented, an attempt should be made to be consistent in sampling from the same position in individuals being compared, and the variability of nematocyst length at each sampled site should be assessed. Inferences can also be made on ontogeny from these data; we conclude that an actinodendrid tentacle grows from the base and at the tips of its branches.     

Comparative ultrastructure of catch tentacles and feeding tentacles in the sea anemone Haliplanella

TEM observations of catch tentacles revealed that the tentacle tip epidermis is filled with two size classes of mature holotrich nematocysts and a gland cell filled with electron-dense vesicles. Vesicle production is restricted to upper-middle and tentacle tip regions, whereas holotrich development occurs in the lower-middle and tentacle base regions. Thus, catch tentacles have a maturity gradient along their length, with mature tissues concentrated at the tentacle tip. Occasional feeding tentacle cnidae (microbasic p-mastigophores and basitrichs) and mucus gland cells occur in proximal portions of catch tentacles, but are phagocytized by amoeboid granulocytes and transported to the gastrodermis for further degradation. No feeding tentacle cnidae or mucus cells occur distally in catch tentacles. Unlike catch tentacles, feeding tentacles are homogeneous in structure along their length with enidocytes containing mature spirocysts, microbasic p-mastigophore or basitrich nematocysts distributed along the epithelial surface. Cnidoblasts are recessed beneath cnidocytes, occurring along the nerve plexus. Mucus gland cells and gland cells filled with electron-dense vesicles are present in feeding tentacles, distributed at the epithelial surface. Granular phagocytes are rare in the feeding tentacle tip, but common in the tentacle base.     

Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system

Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+). We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium. Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx. Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA. The compounds inhibited uptake by the bacterial S. typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain. Cation hexaammines also inhibited growth of S. typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system. In contrast, hexacyano-cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake. The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase. These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation.     

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa,Cnidaria)

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.     

Nematocyst composition of the cubomedusan Chiropsalmus quadrigatuschanges with growth

The nematocysts of Chiropsalmus quadrigatus (Cubozoa; Cubomedusa; Chirodropidae) were examined to determine if their composition changes with an increase in body size. Fixed tentacles of specimens collected in Okinawa, Japan, were homogenized and their nematocysts were observed under a differential interference contrast microscope. Six nematocyst types were observed in medusae of all sizes microbasic mastigophores (MM), large and small trirhopaloids (lTR and sTR), holotrichous isorhizas (HI), ellipsoidal isorhizas (eI), and ovoid isorhizas (oI). Two other nematocysts, large ovoid isorhizas (loI) and microbasic euryteles (ME), were observed only in small individuals. There was also marked difference in proportion of tentacular nematocysts between small and large individuals. HI was the dominant type in small specimens, while MM and eI were predominant in large specimens. Nematocyst composition in the bell and pedalia also differed between small and large individuals. Bells of small medusae contained oI and sTR, while only oI were observed in most large individuals. The pedalia of small medusae had clusters of MM, ME, sTR, and oI. Such single clusters on pedalium bases were characteristic of small individuals. The pedalia of large individuals contained scattered oI. Tentacles of medusae are used for prey capture, so the changes in the major type of nematocysts in tentacles may reflect changes in prey type.     

Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.     

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.     

Competition for space among sessile marine invertebrates: changes in HSP70 expression in two Pacific cnidarians.

The role of stress proteins-either constitutive (HSC) or inducible (HSP)-of the HSP70 family in intra- and interspecific competition for space was examined in two sessile Pacific cnidarians. Anthopleura elegantissima, an intertidal anemone, and Corynactis californica, a subtidal corallimorpharian, express HSP70 in the absence of apparent physical stress. HSP70 protein expression is concentrated in the tentacles of A. elegantissima when the animal is exposed to contact with other benthic organisms. Under the same conditions, however, HSP concentrations are similar in the body and tentacles of C. californica. When two different clones of A. elegantissima interact in the field, the outside polyps (warriors) express more HSP70 than the inside ones (2.4 versus 0.6 ng HSP70/microg Protein). When different C. californica clones interact, HSP70 expression in the outside and inside polyps is similar (1.5 versus 1.8 ng HSP70/microg P) and is fairly constant in the corallimorpharian in the different interspecific encounters. HSP70 expression is related to the different kinds of aggression encountered by both cnidarians. HSP70 expression may be involved in the recovery of tissues damaged by the allelochemical, cytotoxical, or corrosive substances produced by different enemies. C. californica clones appear prepared for war, as evidenced by the high constant expression of HSP70 in the polyps. A. elegantissima exhibits differential HSP70 expression depending on the identity of each neighboring intra- or interspecific sessile competitor. We propose that stress proteins can be used to quantify space competition or aggression among sessile marine invertebrates.     

Cytological Studies of the Nematocysts of Hydra : I. Desmonemes, Isorhizas, Cnidocils, and Supporting Structures

Entire hydras or tentacles were fixed in OsO₄ or in KMnO₄ and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.