Protein Patterns and Synthetic Profiles during Distal Regeneration in Hydra oligactis

Protein patterns and synthetic profiles were examined during distal regeneration in Hydraoligactis . Electrophoretic and radioactive tracer analyses revealed qualitative changes in the general protein profile during regeneration, with a heightened period of protein synthesis between 27–30 hr of regeneration, immediately preceding emergence of the first pair of tentacles. Following this, an increase in collagen-rich mesogleal protein secretion was observed coincident with tentacle initiation. Inhibition of collagen secretion with the proline analog L-azetidine-2-carboxylic acid (LACA) inhibited tentacle formation, and resulted in the development of unique "hypostome buds" at the distal regeneration surface. At the cellular level LACA did not inhibit the nerve cell differentiation that normally precedes tentacle growth, although some predicted decline in cnidocyte production was noted. It is proposed that mesogleal collagen secretion and structural organization may play a major role in the mechanical aspects of Hydra tentacle morphogenesis.     

Analysis of a foot regeneration deficient strain of Hydra oligactis.

A foot regeneration deficient strain of Hydra oligactis with altered size regulation was investigated. Analysis of the concentration of four morphogenetically active substances in Hydra oligactis showed that the foot regeneration deficiency was mainly due to a drastically reduced foot activator concentration. Foot activator was distributed as a very steep gradient in Hydra oligactis leading to a more severe impairment of foot regeneration the closer to the head cutting was done. The concentration of foot inhibitor was comparable, that of head activator slightly increased and that of head inhibitor reduced compared to Hydra vulgaris. Studies at the cellular level implied that the enlarged gastric region was due to an elevated level of inhibition hinting at a shift in the ratio between bound and free head inhibitor in this animal.     

Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

Cell lineages in Hydra: isolation and characterization of an interstitial stem cell restricted to egg production in Hydra oligactis

In an attempt to isolate unipotent stem cells (progenitors to the nerve cells, nematocytes, gland cells, and gametes) from Hydra oligactis females, animals were treated with a drug (hydroxyurea, HU) that preferentially lowers or eliminates the interstitial stem cells, leaving the epithelial tissue intact. In this epithelial environment, interstitial cells remaining after treatment will proliferate and differentiate, permitting a long-term analysis of their developmental capabilities. Following treatment of females with HU, animals were isolated that contained interstitial cells that gave rise to eggs only. Two clones of animals containing these cells were propagated for several years and the growth and differentiation behavior of the interstitial cells examined in their asexually produced offspring. During this time, the cells displayed an extensive proliferative capacity (classifying them as stem cells) and remained restricted to egg differentiation. It is proposed that both the sperm- and the egg-restricted stem cells arise from a multipotent stem cell, which also gives rise to the somatic cells (see above), and that, in hydra, sex is ultimately determined by interactions between cells of the two germ cell lineages.     

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.     

Peptide-driven control of somersaulting in Hydra vulgaris

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Spermatogenesis in Hydra oligactis. II. How temperature controls the reciprocity of sexual and asexual reproduction

Hydra oligactis undergo two mutually exclusive modes of reproduction: at warm temperatures (18-22 degrees C) animals reproduce asexually by budding, while at cold temperatures (10-12 degrees C) gamete differentiation occurs. Using a monoclonal antibody which is specific for cells of the sperm lineage, it was discovered that under conditions where sperm differentiation does not occur (18-22 degrees C), cells continually enter the sperm pathway but progression down the pathway is prematurely halted, effectively blocking the production of sperm. To elucidate the mechanism by which completion of sperm differentiation is controlled, the cell cycle times of interstitial cells entering the sperm pathway at both the restrictive (18 degrees C) and permissive (10 degrees C) temperatures were examined. It was envisaged that at the restrictive temperature the cell cycle times of committed cells would lengthen as they proceeded down the pathway, leading to dilution and eventual loss of cells at later stages of sperm differentiation. This did not occur. Although cells of the sperm lineage were found overall to divide more slowly at 18 degrees C than at 10 degrees C, at both temperatures the cell cycle times shortened as cells proceeded further down the pathway, making a dilution mechanism untenable. The effect of high temperature on the survival of cells was then tested by subjecting animals to a heat shock. Within 12 hr of the increase in temperature, the total number of sperm lineage interstitial cells dropped 10-fold while the total numbers of epithelial and somatic interstitial cells remained virtually unchanged. A distinct consequence of this cell loss was the disappearance of cells furthest down the sperm pathway. It is proposed that as cells move down the sperm pathway, they become increasingly sensitive to high temperature which adversely affects their survival; the higher the temperature, the earlier in the pathway cells die. The lethal effect is abolished by lowering the temperature, allowing sperm differentiation to continue to completion. The possible adaptive advantages of temperature controlling gametogenesis are discussed.     

Calcium- and voltage-dependence of nematocyst discharge in Hydra vulgaris

In Hydra vulgaris, discharge of stenotele nematocysts was induced by contact with prey, electrical stimuli, or increase in the external potassium concentration. In each case 10^-4 M calcium was required in the culture medium. The results indicated a voltage- and calcium-dependent mechanism different from mechano- or chemoreception allowing calcium influx from the external medium. A threshold for activation was suggested by the steep increase of the rate of electrically induced discharge in external fields of 3.5 kV/m. Although organic antagonists for vertebrate calcium channels were ineffective in blocking the calcium-induced nematocyst discharge, inorganic divalent and trivalent cations competitively inhibited the process, with a sequence (Co²⁺ < Ni²⁺ < Cd²⁺ < La³⁺ < Gd³⁺) similar to that seen for antagonism of calcium influx through voltage-dependent channels. Magnesium, an intracellular calcium antagonist, decreased nematocyst discharge, while strontium replacing calcium supported the discharge at a lowered rate. It is concluded that in the nematocyte a voltage-activated influx of calcium through apical ion channels initiates the discharge of the nematocyst in an exocytotic process.     

Sex determination in hydra: control by a subpopulation of interstitial cells in Hydra oligactis males

The stability of sexual phenotype was examined in a single clone of Hydra oligactis males maintained at two culture temperatures, 18 and 22 degrees C. At these temperatures animals of this species do not reproduce sexually, but reproduce asexually by budding, and males and females are morphologically indistinguishable. When the temperature is lowered to 10 degrees C gametogenesis is induced and sexual phenotype can be assayed. Males cultured for several years at 18 degrees C expressed a stable sexual phenotype when induced to undergo gametogenesis; males remained male. Those cultured at 22 degrees C for 1 year, however, expressed a low frequency of sex reversal from male to female; males ceased sperm differentiation and began producing eggs. Male sex reversal in cultures maintained at the higher temperature was correlated with the loss of a specific subpopulation of interstitial cells, those that bind the monoclonal antibody, AC2, which labels cells specific to the spermatogenic pathway in H. oligactis males. When interstitial cells restricted to this pathway were reintroduced into sex-reversed males (phenotypic females), the male phenotype was reestablished and animals reverted to sperm production. To further investigate the role of AC2+ cells in the masculinization of females, normal males (containing AC2+ cells) and sex-reversed males (lacking AC2+ cells) were grafted to females. In grafts between normal males and females, egg production ceased and sperm differentiation ensued, whereas those between sex-reversed males and females continued to produce eggs. Thus, the presence of AC2+ interstitial cells is strictly correlated with male sexual phenotypes and it is only in their absence that the female phenotype is expressed.     

Rapid exocytosis of stenotele nematocysts in Hydra vulgaris

Each cnidarian nematocyte includes a vesicular organelle, called nematocyst, which discharges its content when the cell receives appropriate stimuli. Extracellular electrical stimuli induced discharge of in situ stenoteletype nematocysts in Hydra vulgaris when the apical membrane of nematocytes was depolarized by about 25 mV or more (threshold). Stimuli hyperpolarizing the apical membrane induced discharge only at high amplitudes, adding about 80 mV or more to the resting membrane potential of the nematocyte (resulting in a voltage that may permeabilize the apical membrane). In order to determine the speed of the initiating (exocytotic) process, the delay between stimulus and a clearly visible sign of discharge (i.e., protrusion of the nematocyst's stylets) was measured using video microscopy with triggered flash illumination. The minimal delay was 330–450 s and 230–350 s for depolarizing and large hyperpolarizing stimuli, respectively. With depolarizing stimuli, all discharges of stenoteles occurred between 330 and 950 s after the stimulus. The deviation was caused by differences in the physiological state of the animals tested rather than by variance in the responsiveness of different stenoteles in the same tentacle.Voltage dependence, short latency and Ca/Mg-antagonism are similar to those characterizing exocytosis of synaptic vesicles. This correspondence suggests that discharge of nematocysts is initiated by a similar exocytotic process preceding the ejection of the nematocyst's content.     

Mapping the Whole-Body Muscle Activity of Hydra vulgaris

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Monoclonal Antibodies to Benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the KD values are in the 10(-9) M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216).     

Life history traits and previous exposure predict resistance to UV irradiation in the freshwater cnidarian Hydra oligactis

Abiotic stress is an important source of mortality for cnidarians and is likely to be a major factor shaping their life histories. In freshwater hydra, the ability to withstand exogenous sources of stress varies between species and populations, but little is known about the factors responsible for this variation. Here, we investigated resistance to UV irradiation in Hydra oligactis, a common temperate freshwater cnidarian. We collected polyps from 12 populations and propagated these asexually under standard conditions in the laboratory to obtain 69 laboratory clonal lines with a total of 324 polyps of different age. We measured the size of polyps and recorded their budding rate. In addition, a subset of animals was exposed to hormetic treatment, where experimental animals received a short, sublethal irradiation 2 d before testing their resistance to a higher dose. We investigated how life history traits (age, size, and budding rate), hormetic treatment, and the interaction between life history traits and hormetic treatment relate to the ability of hydra polyps to tolerate high doses of UV irradiation. In multivariate models controlling for the effect of other variables, stress tolerance was positively related to age (lower tolerance in freshly detached buds compared to adult hydra) and size (higher tolerance in polyps with a large body column). Budding rate was negatively associated with stress tolerance. Hormetic treatment increased resistance to UV irradiation, but we found no evidence for an interaction between hormetic response and any of the life history traits, suggesting that the ability to upregulate physiological defense mechanisms after exposure to mild stress does not depend on the life history background of the individuals.     

单克隆抗体的纯化

 <正> 作者采用Bio-Rad公司的单克隆抗体(MoAb)纯化仪(MAPS-100),对两种不同亚类的MoAbs进行了分离纯化,其纯度及免疫学活性均较满意。     

Distinct cellular expression pattern of annexins in Hydra vulgaris

The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.