The effect of glycerol on the Chlorella symbiosis in Hydra viridis

In green hydra strains that are bleached by glycerol, photosynthesis is arrested in both intact hydra and freshly extracted algae whereas photosynthesis is not affected by glycerol in resistant hydra strains and their algae. Glycerol sensitivity is an inherent property of the algae and sensitivity can be transferred to resistant aposymbiotic hydra by infecting them with sensitive algae. It is suggested that the host hydra recognizes glycerol induced changes, other than photosynthetic incompetance, in the algae and either ejects or digests them.Australian Institute of Marine Science, P.M.B. No. 3, Townsville M.S.O. 4810 Australia     

Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis

Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatase nor ferritin was delivered via lysosomes into phagosomes containing live symbionts, whereas these lysosomal markers were found in 50% of the vacuoles containing heat-killed symbionts 1 h after phagocytosis. Treatment of symbiotic algae before phagocytosis with polycationic polypeptides abolishes algal persistence and perturbs the ability of these algae to control the release of photosynthate in vitro. Similarly, inhibition of photosynthesis and hence of the release of photosynthetic products as a result of prolonged darkness and 3-(3,4- dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment also abolishes persistence. Symbiotic algae are not only protected from host digestive attack but are also selectively transported within host cells, moving from the apical site of phagocytosis to a basal position of permanent residence. This process too is disrupted by polycationic polypeptides, DCMU and darkness. Both algal persistence and transport may, therefore, be a function of the release of products from living, photosynthesizing symbionts. Vinblastine treatment of host animals blocked the movement of algae within host cells but did not perturb algal persistence: algal persistence and the transport of algae may be initiated by the same signal, but they are not interdependent processes.     

Bipolar Inhibitory Gradients' Influence on the Budding Region of Hydra viridis

A head inhibitory gradient arising in the vicinity of a hydra'shead, and a foot inhibitory gradient arising from the animal'sfoot are thought to intersect in the budding region. To investigatethe possible influences of these gradients on budding, sampleswere prepared of 25 or more animals having as many as five gastricregions or gastric-plus-budding regions grafted in tandem. Thesegments of grafted animals were considered equivalent exceptfor distance from the head and foot. The average frequenciesof regeneration of budding regions on grafted gastric regions,and the average number of buds on grafted budding regions weredetermined for each sample. Both of these variables changed as functions of distance fromthe head in U-shaped curves as the hypothesis predicts. Thearm of the U closest to the head indicates a stronger degreeof inhibition than the arm closest to the foot since there areless regeneration and fewer buds close to the head. Evidencefor segments on which ectopic heads regenerated also shows inhibitionof budding due to a head. The slopes of the arms of the U-shapedcurves do not resemble the slopes of the curves for head andfor foot inhibition, however, particularly since the slopesfor budding inhibition do not decrease as a function of thenumber of grafted segments. It is doubtful, therefore, thatthe observed bipolar inhibition of the budding region is dueto the inhibitors of head and foot formation.     

Delay in migration of symbiotic algae in Hydra viridis by inhibitors of microtubule protein polymerization

An English strain of the fresh water symbiotic coelenterate Hydra viridis was experimentally "bleached" of its Chlorella algae and maintained indefinitely by feeding. The algal symbiosis could be re-established by injecting other symbiotic algae into aposymbionts. Although algal uptake and recognition were not affected by microtubule protein polymerization inhibitors, these compounds i.e., podophyllotoxin, beta-peltatin and vinblastine had delaying effects on the migration of the algae through the host digestive cells. Picropodophyllotoxin did not delay migration. The rates, the reversibility and the sensitivity of algal migration to low concentrations of drugs known to bind tubulin suggests the symbionts migrate somehow via labile polymerization of host hydra tubulin into microtubules.     

The fine structure of differentiating interstitial cells in Hydra

Summary Interstitial cells of hydra are small undifferentiated cells containing an abundance of free ribosomes and few other cytoplasmic organelles. They are capable of differentiating into epitheliomuscular, digestive, glandular, nerve cells, and cnidoblasts. Developing epitheliomuscular and digestive cells acquire bundles of filaments, 50 Å in diameter, which later are incorporated into the muscular processes. Early gland cells develop an elaborate rough-surfaced endoplasmic reticulum and one or more Golgi apparatus. Secretory granules originate in the Golgi region eventually filling the apex of the cell. Neurons are recognized first by the presence of an elaborate Golgi apparatus, absence of a well-developed endoplasmic reticulum, and later the appearance of cytoplasmic processes. The most striking feature of nematocyst formation by cnidoblasts is the presence of a complex distribution system between protein synthesizing rough-surfaced endoplasmic reticulum and the nematocyst. This system consists of connections between cisternae of the endoplasmic reticulum with smooth Golgi vesicles which in turn are connected to minute tubules, 200 Å in diameter. The tubules extend from the Golgi region around the nematocyst finally entering the limiting membrane of the nematocyst. It is suggested that the interstitial cells of hydra represent a model system for the investigation of many aspects of cell differentiation.This work was supported by grants from the National Cancer Institute (TlCA-5055) and from the National Institute of Arthritis and Metabolic Diseases (AM-03688), National Institutes of Health, Department of Health, Education and Welfare.The author is indebted to Dr. Russell J. Barrnett for his guidance and interest throughout this investigation.     

The general occurrence of intramitochondrial crystals in Hydra cells

Summary Intramitochondrial crystals are found in normal Hydra as well as in animals undergoing various conditions (budding, regenerating, eserinetreated, and sexual). They appear in all regions of the animal, but seem to be more prevalent at or near the extremities: hypostome, tentacles and basal disk. They are found in all of the seven basic cell types: interstitial, cnidocyte, nerve, epithelio-muscular, gland, mucous and digestive cells. The chemical nature of the intramitochondrial crystals is unknown and their significance remains speculative.This investigation was supported by the National Science Foundation, Grant Number Gb-27395     

Effects of Altered Light and Feeding Regimes on the Zooplankton Feeding Capability of Hydra viridis

The effects of light regime, feeding regime and tentacle number on the zooplankton feeding capability of Hydra viridis were tested in the laboratory. Feeding was measured by exposing Hydra to a known volume of Artemia salina nauplii and recording the number captured and ingested. In all cases there was a correlation between the number of Artemia captured and the number ingested. H. viridis with 7 tentacles captured and ingested more Artemia than Hydra with 6 tentacles. However, changes in light and/or feeding regimes did not alter the number of tentacles/Hydra. Varying light and feeding regimes altered the number of Zoochlorellae/cell and Hydra growth rate. There was no effect on the number of Artemia captured or ingested and no effect on the percent ingestion of captured Artemia. These data suggest that, under these conditions, zooplankton feeding by H. viridis is independent of nutritional history.     

The origin of mucous cells inHydra viridis

Summary The origin of hypostomal mucous cells during regeneration and budding has been studied inHydra viridis. NormalHydrae were transected at two levels along their body column — sub-hypostomal and mid-gastric — and the cells which participated in hypostome regeneration were identified histologically and with the electron microscope. An earlier paper in this series (Rose and Burnett, 1968b) showed that zymogen cells transformed to mucous cells in sub-hypostomal regenerates. The work reported in the present paper demonstrates that gastrodermal basophilic cells are the primary source of new mucous cells in animals cut in the mid-gastric region. Evidence is presented to support the thesis that these basophilic cells are derived from epidermal interstitial cells. This choice between zymogen cell versus basophilic cell reflects the distribution of these cells along the parent body column at the sites of the transections.Bud morphogenesis inHydra viridis was also studied because budding provides conditions similar to those of regeneration, and yet, no injury is inflicted on the animal to be studied—that is, a new hypostome must be formed at the distal end of the bud and it must be populated with new mucous cells. The origin of these cells is not from pre-existing mucous cells. The results supported the conclusion that interstitial cells migrate from the epidermis into the gastrodermis of the developing hypostome and differentiate into mucous secretory cells.

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Zusammenfassung Die Herkunft hypostomaler Schleimzellen inHydra viridis wurde während der Regeneration und Knospung studiert. Normale Hydren wurden auf zwei Niveaus ihrer Körpersäule durchschnitten — subhypostomal und mittelgastral — und dann wurden die Zellen, die an der Hypostomregeneration teilnahmen, histologisch und elektronenmikroskopisch identifiziert. Eine frühere Publikation dieser Serie (Rose und Burnett, 1968b) zeigte, daß Zymogenzellen in subhypostomalen Regeneraten sich in Schleimzellen transformierten. Die vorliegende Arbeit demonstriert, daß gastrodermale, basophile Zellen die hauptsächliche Quelle neuer Schleimzellen sind in Tieren, die in der mittel-gastralen Region durchschnitten wurden. Diese basophilen Zellen scheinen von epidermalen Interstitialzellen abgeleitet zu sein. Diese Wahl zwischen Zymogenzellen, resp. basophilen Zellen spiegelt die Verteilung dieser Zellen längs der elterlichen Körpersäule an den Stellen der Schnitte wieder.Knospen-Morphogenese inHydra viridis wurde ebenfalls studiert, weil Knospung ähnliche Bedingungen schafft wie Regeneration, wobei allerdings das Tier keine Verwundung erhält; d. h. ein neues Hypostom muß gebildet werden am distalen Ende der Knospe, und es muß mit neuen Schleimzellen bevölkert werden. Diese Zellen stammen nicht von prä-existierenden Schleimzellen ab. Die Ergebnisse unterstützen die Schlußfolgerung, daß Interstitialzellen aus der Epidermis in die Gastrodermis des sich entwickelnden Hypostoms wandern und sich in Schleimzellen differenzieren.

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Part of a dissertation submitted to the faculty of the Graduate School of Arts & Sciences of Case Western Reserve University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy, 1969.

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Research supported by a grant from the National Science Foundation, No. GB-7345 to A.L.B.     

Excretion of Sugars by Chlorella Species Capable and Incapable of Symbiosis with Hydra viridis

The excretion of some sugars (maltose, glucose, and glucose-6-phosphate) was studied at pH 2.5–6.0 in 38 strains of Chlorella belonging to 15 species of which 7 are capable and 8 incapable of symbiosis with Hydra viridis. A high rate of maltose excretion below pH 4.0 (Cernichiari et al., 1969) was found only in C. vulgaris (non-symbiotic) and C. mirabilis (non-symbiotic). The other Chlorella species are characterized by quite different patterns of sugar excretion. C. spec. (= “C. paramecii”; symbiotic) excretes very high amounts of maltose in the whole range from pH 2.5–6.0. C. kessleri (symbiotic), C. luteoviridis (symbiotic), and C. fusca var. fusca (non-symbiotic) show a predominant excretion of glucose-6-phosphate from pH 2.5–6.0. Some strains also exhibit a high excretion of glucose above pH 4.0 (C. spec. = “C. paramecii”) or below pH 3.0 (C. fusca var. vacuolata). Several species, e.g. C. saccharophila var. saccharophila (symbiotic), C. sorokiniana (non-symbiotic), and C. protothecoides (symbiotic), excrete only very small amounts of sugars. There is no obvious correlation between sugar excretion and the ability or inability of the Chlorella species to form stable symbioses with Hydra viridis.     

The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.     

Distribution of interstitial stem cells in Hydra

The distribution of interstitial stem cells along the Hydra body column was determined using a simplified cloning assay. The assay measures stem cells as clone-forming units (CFU) in aggregates of nitrogen mustard inactivated Hydra tissue. The concentration of stem cells in the gastric region was uniform at about 0.02 CFU/epithelial cell. In both the hypostome and basal disk the concentration was 20-fold lower. A decrease in the ratio of stem cells to committed nerve and nematocyte precursors was correlated with the decrease in stem cell concentration in both hypostome and basal disk. The ratio of stem cells to committed precursors is a sensitive indicator of the rate of self-renewal in the stem cell population. From the ratio it can be estimated that <10% of stem cells self-renew in the hypostome and basal disk compared to 60% in the gastric region. Thus, the results provide an explanation for the observed depletion of stem cells in these regions. The results also suggest that differentiation and self-renewal compete for the same stem cell population.     

The TACE zymogen

The tumor necrosis factor-α-converting enzyme (TACE) is a member of the disintegrin family of metallopro-teinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE’s zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved “cysteine switch” consensus motif within TACE’s Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE’s susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB-3CT. These findings point at the possibility of developing, in the near future, a new generation of anti-inflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers.