Androgen metabolism in somatic and germinal tissues of the sea star Asterias vulgaris.

1. Cell-free homogenates of male and female pyloric caeca, body wall, testis and ovary were incubated with radiolabeled 3H-androstenedione. 2. Pyloric caeca had highest rates of androstenedione conversion. The predominant metabolites in the pyloric caeca were testosterone, 5 alpha-androstane-3 beta, 17 beta-diol and 5 beta-androstane-3 beta, 17 beta-diol. 3. In body wall, testicular and ovarian homogenates, androstenedione was converted primarily to testosterone and also to 5 alpha-androstanedione and epiandrosterone. 4. Qualitative and quantitative differences in androgen metabolism in somatic and germinal tissues may be related to tissue-specific regulation of cellular metabolism.     

Photoperiodic regulation of gametogenesis in a North Atlantic sea star,Asterias vulgaris

Summary

Individuals of the sea star Asterias vulgaris from New Hampshire were maintained under two contrasting regimes of seasonally changing photoperiod. In one regime the lights turned on and off in phase with local sunrise and sunset; in the other, the daily photoperiod was kept 6 months out of phase with ambient photo- period. When dissected after being maintained for 18 months, the gametogenic condition of the out-of-phase animals was found to be 6 months out of phase with that of the in-phase animals. This experiment demonstrates that photoperiodic regulation of reproduction is important even in marine animals that normally experience marked seasonal changes in sea temperature.     

Control of ornithine decarboxylase activity in alpha-difluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues

The regulation of ornithine decarboxylase (ODC) activity by the polyamine derivatives N1,N8-bis(ethyl)-spermidine and N1,N12-bis(ethyl)spermine was studied using a line of L1210 cells resistant to alpha-difluoromethylornithine (D-R cells), which contain very high levels of ODC, and a synthetic mRNA prepared from a plasmid containing an insert corresponding to ODC mRNA adjacent to an SP6 RNA polymerase promoter. Studies in which ODC protein was labeled in the D-R cells by exposure to [35S]methionine indicated that the polyamine derivatives and their physiological counterparts led to an increased rate of degradation of ODC and to a rapid reduction in ODC synthesis without affecting the content of ODC mRNA. Direct evidence that the polyamine derivatives act by inhibiting the translation of the ODC mRNA was obtained by studying their effects on the translation of ODC mRNA in reticulocyte lysates. This translation was strongly inhibited by the addition of N1,N8-bis(ethyl)spermidine, spermidine, N1,N12-bis(ethyl)spermine, or spermine but was not affected much by putrescine. The inhibition of the translation of ODC mRNA by either of the bis(ethyl) polyamine derivatives occurred at concentrations which stimulated total protein synthesis showing the selectivity of the reduction in ODC. The effects of polyamine derivatives and polyamines on translation of the plasmid-derived ODC mRNA were identical with those found with the D-R L1210 cell mRNA. This synthetic ODC mRNA lacks 261 bases of the 5'-leader sequences and 200 bases plus the poly(A) section from the 3'-nontranslated sequence. Therefore, these regions appear not to influence sensitivity of the ODC mRNA to inhibition of translation by polyamine derivatives.     

Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

Ornithine decarboxylase activity and polyamines in relation to aging of human fibroblasts

A reduction of ornithine decarboxylase (ODC) activity is associated with the decreased growth rate in early senescence of human embryonic lung fibroblasts. Inhibition of ODC activity with α-methyl ornithine in younger cells reduces cell proliferation; inhibition of putrescine deamination with aminoguanidine increases intracellular putrescine concentration and stimulates cell proliferation. The data suggests that the decreased cell proliferation which occurs in senescence may, at least in part, be dependent upon the reduced ODC activity.     

Effect of plant growth hormones and polyamines on ornithine decarboxylase activity during the germination of barley seeds

Gibberellic acid (GA₃) and β-indolylacetic acid (IAA), two of the well known growth hormones, induce four fold the activity of ornithine decarboxylase (ODC) during the germination of barley seeds ( Hordeum vulgare L. var. Beca). The optimal concentration for induction of ODC was 10^–5 M for GA₃ and 10^–3 M for IAA. When 10^–3 M of a polyamine, putrescine or spermidine, is added to the growth medium, ODC activity is significantly inhibited. This inhibition is due to the induction of a protein inhibitor of ODC (antizyme), whose apparent molecular weight is 16 000 ± 2 000 daltons. Addition of GA₃ to cultures which have been grown for 50 or 98 h in the presence of polyamines, abolishes the observed inhibition of ODC activity, while in the reverse experiment, addition of polyamines at 50 or 98 h does not affect the ODC activity induced by GA₃. Cadaverine, a physiological plant diamine, enhances ODC activity; whereas 1,8-diaminooctane (the alkyl analogue of spermidine) does not have any effect.     

Regional changes in ornithine decarboxylase activity and polyamine levels during thyroxine-induced cardiac hypertrophy

Ornithine decarboxylase activities (ODC) and polyamine levels were determined in five cardiac regions of the rat heart, following daily administration of 1 mg/kg of thyroxine, in the right and left atria, the right and left ventricles and the septum. The thyroxine stimulated ODC activity in all five regions of the heart. Enzyme activity in the left atrium and the septum peaked a day earlier than in other regions and the decline of ODC activity was slower. Putrescine in control animals was present in all regions except the right atrium, where its content was below detectable levels. Following the administration of thyroxine, the putrescine content of the left atrium, the right ventricle and the septum declined, while spermidine and spermine levels remained unchanged. In direct contrast to the other regions of the heart, thyroxine stimulated an increase in polyamines, as well as in weight which occurred exclusively in the left ventricle. These findings suggest a causal relationship between increased polyamines and hypertrophy.     

Occurrence of ornithine decarboxylase and polyamines in cartilage.

The activity of ornithine decarboxylase was investigated in cartilage from chick embryos, rabbits, rats and human foetuses. The enzyme activity in these cartilages was of the same order as the detected in other body tissues. Ornithine decarboxylase activity in chick-embryo cartilage and liver was the same when compared on the basis of total soluble tissue protein. The cartilage enzyme exhibited a pH optimum of 6.5 and a Km for ornithine of 0.16mM. Ornithine decarboxylase activity in chick-embryo pelvic leaflets was maintained at the value in vivo for up to 22h when the isolated tissue was incubated in a modified Waymouth's medium (MB 752/1) at 37 degrees C. After addition of cycloheximide to the incubation medium, ornithine decarboxylase activity declined, with a half-life of 40 min. The concentrations of the polyamines spermidine and spermine in chick-embryo pelvic cartilage and rabbit costal cartilage were of the same order as the concentrations detected in other tissues.     

Changes in ornithine decarboxylase activity and polyamine levels during the growth of Tetrahymena thermophila cultures

Ornithine decarboxylase activity and polyamine levels were determined at various growth phases of Tetrahymena thermophila cultures. Enzyme activity and intracellular polyamines increased in exponentially growing cells and peaked just before the stationary phase. Putrescine was the predominant polyamine and spermidine and spermine concentrations were low throughout. The increase in putrescine level can be totally accounted for by the enzyme activity detected, provided that there is an ample supply of the precursor, L-ornithine.     

Ultrastructure of spermatogenesis in the sea star,Asterina minor

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron-dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi-derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron-dense material is deposited under the electron-dense disk; and (6) the nucleus forms a hollow opposite the electron-dense material.     

Progesterone and oestrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: A comparison with the corresponding levels in the female sea star

• 1.1. Levels of progesterone and oestrone were estimated by means of RIA in the gonads and pyloric caeca of male specimens of Asterias rubens throughout the reproductive cycle.
• 2.2. The patterns obtained appeared to be sex-specific on comparison with those of female specimens of A. rubens and it was concluded that progesterone and oestrone in the testes are involved in the regulation of gametogenesis.
• 3.3. The levels of progesterone in the pyloric caeca were about ten times higher than those in the testes; in the same sequence levels of oestrone were only slightly higher.
• 4.4. The onset of a new reproductive cycle, taking the abrupt decrease of the pyloric caeca-index as a marker, coincides with a strong decrease of the progesterone level (P < 0.001) and an increase of the oestrone level (P < 0.01) in the pyloric caeca.
• 5.5. It is supposed that progesterone and oestrone in the pyloric caeca are concerned with making available the materials required for gametogenesis.

     

Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite-resistant Leishmania

The levels of trypanothione, a glutathione-spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene amplification in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene amplification. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is amplified in the mutants. The co-transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.     

Fate of ingested leucine and glucose in the sea star,Asterias rubens,during its annual reproductive cycle

• 1.1. The distribution of radiolabel from L-leucine [¹⁴C-UL] and D-glucose [¹⁴C-UL] was measured in the sea star Asterias rubens at 1, 6 and 24 hr after oral administration.
• 2.2. Incorporation of the label from both compounds was observed in pyloric caeca, coelomocytes and ovaries even after an incubation time of 1 hr.
• 3.3. Highest incorporation from both precursors was found in proteins, while substantial radioactivity was present in the amino acids, organic acids and neutral components. Lipids were hardly labelled from leucine and only slightly from glucose.
• 4.4. Radioactivity in proteins and lipids increased with increasing incubation time. No significant differences were found in the distribution patterns of radiolabel during the reproductive cycle.
• 5.5. The data obtained are discussed in terms of current knowledge on the translocation of nutrients in echinoderms.

     

Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.     

Learning of inhibition of behavior in the sea star Asterias rubens

This work presents results of study of a peculiar form of behavior of the sea stars Asterias rubens, which can be called transient from the unconditional-reflex reaction of the standing still to action of the an outside stimulus or unfavorable environment factor to the elaborated internal inhibition of the higher animals. The experiment was based on use of the biological peculiarity of behavior of sea stars (placed in a vessel)—a congenital reflex of movement to the water surface. At repeated meeting with the unfavorable action (the water surface desalting or a tapping on the upper rays), the sea star descends to the vessel bottom, is firmly attached with ambulacral feet to the vessel wall and stops rising to the surface. This state can be disinhibited by throwing the sea star onto the bottom. After a long training (for several experimental days) the sea stars begin to crawl on the bottom without ascending to the water surface. On destruction of the nervous system intactness (two rays with the madrepore plate were cut) the reflexes of the rise to the surface and escape of the unfavorable factors are preserved, but the active cessation of the rise does not appear. The observed phenomenon is similar to the elaboration of the reflex with negative reinforcement in the higher animals, at which there occurs inhibition of the congenital reaction that is inadequate under changed environment conditions. At present, neurophysiological mechanisms of elaboration of such “forbidden” inhibition are unknown.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Calcium flux and ornithine decarboxylase activity in cultured endothelial cells

This brief communication reports the observation that calcium influx appears to be a requirement in the serum-induction of ornithine decarboxylase (ODC) activity in cultured aortic endothelial cells. Addition of 35% fetal calf serum causes an increase in endothelial ODC activity within three hours to levels that are 16 times those of baseline. Preincubation of EC with lanthanum chloride (LaCl) or the addition of ethylene glycol (β-aminoethyl ether)-N-N′ tetraacetic acid (EGTA) to the medium inhibits the serum-induction of ODC. The displacement of the lanthanum ions with EGTA reverses the inhibition which demonstrates the viability of the LaCl₃-pretreated cells, and lends support to the view that calcium may be involved in the induction of ODC.     

Effects of a high soy protein diet on intestinal polyamines and ornithine decarboxylase activity in rats

This study was performed to determine whether intestinal luminal polyamine concentrations are affected by a high soy protein diet when compared with a high casein diet or a normoprotein casein diet. We also determined the effects of these diets, with differences in polyamines content, on mucosal polyamines and ornithine decarboxylase (ODC) activity to assess cell proliferation. Three groups of eight male Wistar rats were fed either a 50% soy protein diet, a 50% casein diet, or an 18% casein diet as a control. After 4 weeks of feeding, both intestinal content and mucosa were recovered. Polyamines were assayed by high performance liquid chromatography. ODC activity was measured by the release of (14)CO(2) from (14)C-L-ornithine. Luminal putrescine and cadaverine concentrations were higher in the jejunum than in the ileum, suggesting an absorptive process. The highest concentrations of intestinal polyamines were observed in rats fed the soy protein diet (P < 0.05). Only minor differences were observed in mucosal polyamines according to the diets. ODC activity was also higher in the intestinal mucosa of rats fed the high soy protein diet (P < 0.05). These results suggest that intestinal luminal polyamine concentrations and ODC activity are modulated by the dietary protein source.