Artemia Salina Embryos Research Articles

Effect of the Choline-Based Ionic Liquids on Cryopreservation of Hydrated Cysts of Artemia Salina Embryos

Effect of the Choline-Based Ionic Liquids on Cryopreservation of Hydrated Cysts of Artemia Salina Embryos

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.

Acetylcholinesterase activity in embryonic and larval development of Artemia salina leach (crustacea phyllopoda)

AbstractAcetylcholinesterase (AChE) activity has been studied in Artemia salina embryos and larvae by quantitative, histochemical, and electrophoretic methods. An AChE activity is present in dehydrated encysted gastrulae and remains low in rehydrated developing embryos, showing at hatching an increase that becomes faster during the nauplius stages (twice the gastrula activity at the nauplius II stage), and continues linearly in the first metanauplius stages. The histochemical staining, faint and diffuse in dehydrated gastrulae, in more advanced embryos becomes localized in cell membranes and stronger in differentiating tissues; most AChE activity is localized in the nervous and muscular cells, and a fainter one in the differentiating metanauplius segments. Dehydrated cysts show one electrophoretic AChE band, that is no longer detectable in rehydrated embryos and larvae, while two other bands appear, that migrate more slowly; in advanced larval stages, the more cathodic band is predominant. In dehydrated cysts, isoelectric focusing separates three AChE bands with isoelectric points (IP) at pH 5.0, 5.7, and 5.9; in hydrated embryos and larvae, the band at pH 5.0 is absent and another is seen at pH 5.5. Eserine, DFP, and BW284c51 inhibit the enzyme activity strongly and cause paralysis of larvae; also tubocurarine blocks the motility. These results are discussed in relation to the differentiation of the neuromuscular system and to the development of motility. As a hypothesis, in early embryonic stages, before any neuromuscular differentiation is evident, a cholinergic‐like system might regulate non‐neural cell activities and interactions, associated with embryogenetic events.

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.

Purification and properties of an elongation factor functionally analogous to bacterial elongation factor Ts from embryos of Artemia salina.

A protein factor which is functionally analogous to bacterial EF‐Ts was purified from developing embryos of the brine shrimp Artemia salina. The factor, designated as eEF‐Ts, has properties which are similar to EF‐1β from pig liver. Specifically eEF‐Ts in our assay conditions stimulates EF‐1L(eEF‐Tu)‐dependent binding of aminoacyl‐tRNA to ribosomes and eEF‐Tu and EF‐2 dependent poly‐phenylalanine synthesis 2.5–3‐fold. In addition eEF‐Ts is an efficient catalyst of a guanine nucleotide exchange reaction involving eEF‐Tu · GDP complexes and free GTP. We suggest that the latter activity explains the stimulation by eEF‐Ts of eEF‐Tu‐dependent functions.eEF‐Ts consists of a single polypeptide chain of Mr 26 000, as judged by electrophoresis on acrylamide gels containing dodecylsulfate. However, the native enzyme consists largely of high‐molecular‐weight aggregates with sizes ranging from about 100 000 to greater than 200 000. Stable complexes of EF‐Tu and eEF‐Ts were formed by combining the two factors and these complexes were capable of binding GTP at low temperature (0–2 °C). Antibodies against the heavy form of elongation factor 1 cross‐reacted strongly with purified eEF‐Ts confirming our previous observation that EF‐1 in Artemia consists of eEF‐Tu · eEF‐Ts complexes. The relationship between EF‐1 and eEF‐Tu and eEF‐Ts is discussed.

α,α-trehalase from the brine shrimp Artemia salina. purification and properties

1. 1. Trehalase (α,α-trehalose 1- d-glycohydrolase, E.C. 3.2.1.28) from cryptobiotic Artemia salina embryos was purified and characterized. 2. 2. Most of the enzyme activity was present in an insoluble form and could be solubilized by deoxycholate treatment at high ionic strength and sonication. 3. 3. The enzyme had a molecular weight of 75,000 and its isoelectric point was 6.2. It was optimally active at pH 5.6 with a K m = 4.3 × 10 −3 M for its natural substrate. 4. 4. The enzyme was highly specific for trehalose, only lactose and cellobiose being hydrolyzed to a limited extent.

Eucaryotic oligonucleotides affecting mRNA translation

High speed-supernatants and ribosomal salt washes of dormant and developing Artemia salina embryos contain a potent inhibitor of translation; it blocks the elongation factor EF-1-dependent ribosomal binding of aminoacyl-tRNA. A translation activator that counteracts the effect of the inhibitor is found in the same fractions from developing embryos; there is little activator in undeveloped cysts. The appearance of the activator may be responsible for the onset of protein synthesis when development resumes. Both compounds are oligonucleotides. The inhibitor, M r about 6000, is rich in pyrimidines (47% U, 11% A, 26% C, 16% G), sensitive to RNase A, and resistant to RNase T1. The activator, M r about 9000, is rich in guanine (33% U, 10% A, 6% C, 51% G), sensitive to RNase T1, and resistant to RNase A. It complexes with the inhibitor and inactivates it. Inhibitor and activator seem to be end products of hydrolysis of embryo RNA by RNase T1 and RNase A, respectively, and ribosomal salt washes of developing embryos have higher RNase A activity than corresponding fractions from dormant cysts.

Disaggregation of elongation factor 1 by extracts of [formula omitted][formula omitted

Extracts of 40 hr Artemia salina nauplii can convert a heavy form of elongation factor 1 (EF-1 H) to a light species (EF-1 L). The data indicate that a protease in the extracts is responsible for this reaction, and these findings may explain the observation that extracts from Artemia salina nauplii have only EF-1 L whereas before hatching of the Artemia salina embryos EF-1 H is the predominant species (Slobin and Mőller [1975] Nature 258, 452–454).

Messenger RNA in undeveloped and developing Artemia salina embryos

Summary Messenger activity of RNA species retained [poly (A)+] and not retained [poly (A)−] by oligo(dT) cellulose was assayed in cytoplasmic fractions of Artemia salina embryos using a wheat germ system. In undeveloped embryos, mRNA activity was found in a fraction sedimenting at 15,000 x g (p15) and about half as much in the cytosol (author05). The ribosome (R) fraction, consisting mainly of 80S monomers, had no activity. Only p15 contained [poly (A)+] mRNA amounting to about one tenth of the total mRNA activity of undeveloped embryos. After development for 15–16 hrs, the total mRNA activity of the embryos increased 3-fold. All three cell fractions contained mRNA activity in both [poly (A)−] and [poly (A)+] RNA. Over half of the total was in the polysome-containing R fraction and about one fifth in [poly (A)+] RNA. The most pronounced change with development was in the [poly (A)+] mRNA activity which increased over 6-fold. The patterns of polypeptides synthesized by translation of [poly (A)+] mRNA fractions from undeveloped and developing embryos were similar but not identical.

Polypeptide chain initiation in eukaryotes: initiation factor MP in Artemia salina embryos.

The activity of IF-MP, a polypeptide chain initiation factor that forms a ternary complex with eukaryotic initiator Met-tRNA and GTP and promotes binding of the initiator to 40S ribosomes, is very low in undeveloped Artemia salina embryos but increases over 20-fold following resumption of development upon hydration of the cysts. The factor is present in both the ribosomal salt wash and high-speed-supernatant. Its specific activity is 50 times higher in the wash but its total activity is only about twice as high in the wash as in the supernatant. As is true of IF-MP from other eukaryotic sources, the A. salina factor is specific for eukaryotic Met-tRNAi and sensitive to SH-reagents, and its activity is GTP dependent.

Effect of development on the translation of messenger RNA in Artemia salina embryos.

Cell-free preparations from encapsulated Artemia salina embryos readily translate poly(U), but there is no endogenous protein synthesis or translation of added natural mRNA until development resumes upon incubation in saline. High-salt-washed 80S ribosomes and highspeed supernatant (cytosol) were prepared from undeveloped eggs and from eggs allowed to develop to a stage prior to hatching. Endogenous protein synthesis occurs only with ribosomes from developed embryos, whether with undeveloped or developed cytosol. This is mainly elongation of preformed polypeptide chains, for it is largely resistant to edeine, an inhibitor of chain initiation. Edeinesensitive translation of added natural mRNA occurs with both developed and undeveloped ribosomes but requires developed cytosol. Translation of brome mosaic virus RNA is not much, if at all, further stimulated by high-saltwash of developed ribosomes, but that of globin mRNA is markedly enhanced. Levels of the chain initiation factor EIF-1 are the same before and after development. These results are consistent with the view that resumption of developemtn is triggered by transcription, with ensuing translation of the resulting messengers to yield, among other proteins, mRNA-recognizing initiation factors of the IF-3 type which are partly free in the cytosol and partly ribosome-bound. The data also suggest that different factors may be involved in the translation of brome mosaic virus RNA and globin mRNA by this system.

The structure of the shell and outer membranes in encysted Artemia salina embryos during cryptobiosis and development

Slices of cryptobiotic and developing cysts of the brine shrimp, A. salina , were fixed in glutaraldehyde and osmium tetroxide for electron microscopy. The shell was similar to insect egg shells in having a hypochlorite-soluble chorion and a fibrous, hypochlorite-resistant cuticle. Between the chorion and cuticle was a thin but complex membrane that resisted solution in hypochlorite and was impermeable to lead ions. The extra-embryonic, subcuticular space usually contained many small particles in the cryptobiotic cyst. The particles disappeared during development and were replaced by the hatching membrane. After being released from the brood sac, at least some cysts are covered by a cellular membrane. Upon air-drying (i.e., entry into cryptobiosis) this outer membrane is destroyed, and subsequent wetting reinitiates development in the encysted gastrula.

The origin of threhalose and its significance during the formation of encysted dormant embryos of Artmia salina

1. 1. A study has been made of trehalose and other carbohydrates in Artemia salina embryos of two types: one which enters dormancy as a blastula and one which undergoes direct development. 2. 2. After 10 hr of development the presumptive dormant embryo contains at least eight times and amount of trehalose present in the non-dormant embryo. When the blastula enters dormanyc trehalose constitutes about 15 per cent of the dry weight, whereas non-dormant embryos of comparable age have essentially no trehalose. 3. 3. Analyses of female hemolymph and studies on presumptive dormant embryos in vitro show that trehalose is snythesized by the developing embryos. 4. 4. The glycogen levels of both types of embryos are similar after 10 hr of development. During subsequent development of the presumptive dormant embryo the glycogen content decreases markedly, but it remains at a high level in the non-dormant embryo. 5. 5. Presumptive dormant embryos contain about twice the amount of glycerol present in non-dormant embryos after 10 hr of development, and this relationship remains about the same with further development. 6. 6. The significance of trehalose accumulation in Artemia embryos is discussed.