Egg Homogenates Research Articles

Isolation of a Polysaccharide Antigen from Schistosoma Mansoni Eggs

Abstract A polysaccharide antigen was isolated from Schistosoma mansoni egg homogenates by the phenol procedure. The crude preparation (CPEA) contained at least two antigens. The more purified antigen (PEA) was isolated by sequential enzymatic treatment of CPEA with DNase, RNase, Pronase, and α-amylase. PEA was resistant to boiling, freezing and thawing, mild acid and alkali, and chloroform, but was destroyed with periodate. It gave a positive reaction with anthrone reagent. PEA was eluted in the wash fraction from a DEAE cellulose column and in the void volume of a Sephacryl 200 column. After immunoelectrophoresis and polyacrylamide electrophoresis there was little or no migration. Amino acid analysis failed to reveal ninhydrin-positive material in the a hydrolyzate of PEA. These results suggested that PEA is a neutral polysaccharide with a m.w. of more than 200,000 and contains no amino acids or hexosamine. Antibodies against PEA were detected in sera obtained from mice infected with S. mansoni. PEA is different from previously described antigens derived from schistosome eggs.

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata.

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.

Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 m M from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 m M αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 m M) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 m M), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 m M) or spermine (0.1–0.8 m M). Especially, spermine (0.5 m M) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 m M).

Adenosine 3′,5′‐cyclic monophosphate‐binding protein in sea urchin eggs and embryos

AbstractAdenosine 3′,5′‐cyclic monophosphate (cAMP) binds to high‐molecular substances which are probably proteins, in homogenates of sea urchin eggs and embryos. The bound cAMP is exchangeable. Optimal pH for the binding capacity of the proteins with cAMP is 4.0, and is shifted to 5.0 in the presence of 5 mM caffeine. The level of bound cAMP increases steeply during 10 minutes of incubation. This is then followed by a less steep increase. The level of bound cAMP decreases in the presence of NaCl. The dissociation onstant between cAMP and the proteins in homogenates of unfertilized and ertilized eggs is about 10 nM, and the value in the embryos at the gastrula stage is lower than that in the unfertilized egg homogenate.

Heat labile activating substance for ornithine decarboxylase activity in sea urchin eggs

AbstractIn sea urchin eggs, the activity of ornithine decarboxylase (ODC) [ E C 4.1.1.17] is detectable only in the particulate fraction yielded by centrifuging egg homogenates at 10,000g for 30 minutes. ODC activity in the particulate fraction isolated from fertilized eggs is higher than that from unferti‐lized eggs. ODC activity in the particulate fraction isolated from either unfertilized or fertilized eggs is enhanced by adding the supernatant fraction obtained by centrifugation at 105,000g for two hours. Heating this supernatant at 70°C for 15 minutes results In complete loss of the stimulating capacity for ODC activity. Sea urchin eggs seem to contain heat labile activating substance(s) for ODC activity. The substance does not pass through the ultrafiltration membrane Diafro UM–10. Only eggs and unhatched embryos, in which mitosis occurs frequently, contain the activating substance. In the presence of the activating substance, Ca2+enhanced ODC activity.

Purification and partial characterization of Culex pipiens fatigans yolk protein

Extraction of washed Culex pipiens fatigens egg homogenate pellets with high ionic strength-high pH medium yields a crude yolk extract which can be divided into three fractions by gel chromatography. The major fraction resulting from chromatography appears to be Culex yolk protein. Our studies suggest that Culex pipiens fatigens yolk protein has a molecular weight of 3.8 × 10 5 daltons and contains protein bound phosphate, lipid and carbohydrate, as do many other vertebrate and invertebrate yolk proteins. Culex yolk protein appears to be composed of two classes of polypeptide chains of molecular weights of 1.6 × 10 5 and 0.82 × 10 5 daltons. Yolk protein appears to aggregate under the conditions used in this investigation.

CHANGE IN THE ACTIVITY OF ARYLESTERASE IN SEA URCHIN EGGS FOLLOWING FERTILIZATION.

The activity of arylcsterase in sea urchin eggs (Anthocidaris craxsispina), increases at 5 min after fertilization to about 1.5-fold that in unfertilized eggs, and decreases at 15 min to a lower level than that in unfertilized eggs. Then the activity of the enzyme increases again. The enzyme activity in unfertilized eggs is enhanced by either fructose 1, 6-diphosphate (FDP) at concentrations between 4 and 10 μM, or guanosine 3', 5'-cyclic monophosphalc (cGMP) at concentrations between 0.1 and 0.3 μM. The activity is detectable in the crude microsomal fraction and also in the supernatant fraction obtained from sea urchin egg homogenates by centrifugation at 105,000 × g for 2 hr.

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO 4 and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.

Lipid metabolism in early development using labeled precursors incorporated during oogenesis and in cell‐free embryo homogenates

Embryos of the toad, Bufo arenarum, Hensel, taken during early stages of development were used to survey the [U-14C]glycerol and 32P lipid labeling. When precursors were supplied at the time of oogenesis, large differences in specific activities of phospholipid were observed. Using 32P, a steep rise as a function of development was evidenced. Triglycerides contained much higher proportions of [U-14C] glycerol than phospholipids when administered to the female toad along with a pituitary homogenate. However, lack of [U-14 C] glycerol uptake into lipids was observed when cell-free homogenates of eggs at different stages of development ranging from unfertilized oocyte to midgastrula were incubated in unsupplemented amphibian Ringer. At the later stage, significant de novo biosynthesis of lipids from glycerol began to be measurable, whereas during cell cleavage intracellular redistribution of preformed phosphoglycerides was used for membrane assembly.

Côntrole neuroendocrine des protéines sanguines vitellogenes d' Anacridium aegyptium sain et parasite

The changing patterns of haemolymph proteins were followed in male and female adults of normal and parasitized Anacridium aegyptium during diapause (autumn, winter) or during activity (spring) of their endocrine system without or with electrostimulations of the pars intercerebralis (PI). The haemolymph protein concentration is high in winter and decreases in spring. It is comparatively depleted in locusts infected by the fly Metacemyia calloti. However, the depletion is significant only in ‘castrated’ females. Fifteen protein fractions were resolved by polyacrylamide disk gel electrophoresis in haemolymph of normal and infected locusts during diapause and activity. Some fractions decrease in quantity during activity in males, normal females, and parasitized females with complete ovarian development. One fraction disappears in females with mature eggs and seems correlated with formation of the eggshell. Eight others protein fractions exhibit electrophoretic mobility identical to the 7 protein fractions of homogenates of eggs. There is little doubt that these haemolymph protein fractions are involved in yolk synthesis and are thus ‘vitellogenic’. One of these ‘vitellogenic’ fractions (band 6) is larger in yolk than in blood. Five protein fractions were demonstrated by electrophoresis of homogenates of parasites. Their electrophoretic mobilities are similar to those of 5 of the 8 haemolymph ‘vitellogenic’ fractions of the host. There is little doubt that these 5 haemolymph protein fractions (one of them is the band 6) are involved in the nutritional requirements of the parasite. Electrostimulation of the PI, during diapause and activity, increase the haemolymph protein concentration and chiefly the protein concentration of the blood band 6. Thus, the median neurosecretory cells of the brain (M-NSC) regulate protein synthesis and chiefly the synthesis of ‘vitellogenic’ proteins. In parasitized females, the increase of the haemolymph protein concentration after electrostimulations of the PI is associated with an enhancement of ovarian development. The depletion of the haemolymph protein concentration in ‘castrated’ females is thus involved in the inability of the oöcytes to sequester available proteins from the haemolymph. The haemolymph protein deficiency may be attributed to (1) an impairment of protein synthesis, attendant upon the hypoactivity of the M-NSC, and (2) the nutritional requirements of the parasite.

REDUCTION OF POLYCHLORINATED BIPHENYLS IN SHRIRAP AND EGGS BY FREEZE‐DRYING TECHNIQUES

ABSTRACTConsidering the toxicity of polychlorinated biphenyls (PCBs), which poses threats to environment and human health, this project was designed to develop food processing techniques which can eliminate and/ or reduce these compounds. The potential of freeze‐drying methods in reducing PCB levels was evaluated using a precontaminated egg and shrimp homogenate. PCBs were recovered and identified by extraction, florisil column‐cleanup, gas chromatography and mass spectrometry. Results indicated 40.3% and 25.2% reduction in shrimp homogenate containing Aroclor 1254 and 1260 respectively. The parallel experiment conducted on whole eggs showed 26.8% and 26.6% reduction respectively, for both the Ardclors. Several factors predicted to be responsible for increased PCB losses in shrimp homogenate compared to whole egg samples are outlined. Since freeze‐drying techniques can be used for these products, they can prove to be of added advantage by reducing toxic PCBs.

Esterases in relation to embryonic diapause in the field cricket, Teleogryllus commodus

Fourteen esterase isozymes were detected by polyacrylamide gel electrophoresis in homogenates of eggs of the field cricket Teleogryllus commodus, at various stages of development. One esterase isozyme appeared during embryonic diapause termination effected by prolonged chilling of the eggs. Esterase activity towards methyl butyrate in T. commodus egg homogenates increased with time. Sonication generated the maximum levels of esterase activity observed. Methyl butyrate esterase activity in T. commodus eggs was high during the first few days of embryogenesis; fell markedly during the period of rapid water uptake, and then remained fairly constant during diapause, diapause development, and during resumed embryogenesis.

Treatment of Salmonella-Arizona-infected turtle eggs with terramycin and chloromycetin by the temperature-differential egg dip method.

Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle.

Heterogeneous distribution of “lysosomal” hydrolases in yolk platelets isolated from unfertilized sea urchin eggs by zonal centrifugation

Three typical “lysosomal” glycosidases, α- L-fucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase, were localized within the yolk platelets of unfertilized Strongylocentrotus purpuratus eggs. Homogenates of eggs were fractionated by rate-zonal centrifugation, and the isolated particles were subjected to integrated biochemical and morphological (electron microscopic) analysis. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1,3 glucanase) in the sucrose density gradient. Yolk platelets were isolated in a high state of purity, with contamination by mitochondria and cortical granules at trace levels. Enzymatic heterogeneity exists within the yolk platelet population. Acid nitrophenyl phosphatase and α- l-fucosidase activities appear to be uniformly distributed within all the yolk platelets, while N-acetyl glucosaminidase and galactosaminidase activities appear to be preferentially distributed within the slower sedimenting sub-population of yolk platelets. Another band of hexosaminidase containing particles sedimented slightly slower than the bulk of the yolk platelets, coincident with the mitochondria. The acid hydrolases packaged in the yolk platelets may participate in the mobilization of yolk material after fertilization. The yolk platelet thus appears to be a highly complex and structured “lysosome-like” storage organelle.

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of 3H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated 3H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization. In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase. Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs.

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.

Treatment of Salmonella-Arizona-Infected Turtle Eggs with Terramycin and Chloromycetin by the Temperature Differential Egg Dip Method

Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle.

Is calcium ionophore a universal activator for unfertilised eggs?

WE have previously exposed sea urchin eggs to micromolar amounts of the divalent transporting ionophore A23187, and observed every parameter of normal fertilisation. The cortical reaction with elevation of the fertilisation membrane, the plasma membrane conductance changes, the respiratory burst, and increases in protein and DNA synthesis were all initiated in the usual fashion1. These activations of Lytechinus pictus and Strongylocentrotus purpuratus eggs were independent of the ionic composition of the external solutions1. A23187 seemed to act by releasing intracellular Ca2+, for eggs preloaded with 45Ca showed a twentyfold increase in 45Ca-efflux when activated by ionophore or fertilisation1. Measurements of free and bound calcium and magnesium in homogenates of unfertilised eggs showed that most of the Mg2+ was already available in soluble form, whereas the Ca2+ was sequestered but available for release1. After we found that 50 µM A23187 could activate eggs of the mollusc Acmaea insessa and the protochordate Ciona intestinalis to undergo several abnormal cleavages, we wondered whether ionophore activation might be a general phenomenon. To our knowledge artificial parthenogenesis has not been obtained with Acmaea and Ciona eggs by other methods. We have now found that calcium ionophore activation is common to several species widely separated phylogenetically.

Peroxidatic activity of catalase in the cortical granules of sea urchin eggs

The presence of peroxidatic activity of catalase in eggs of the sea urchins Hemicentrotus pulcherrimus and Temnopleurus toreumaticus was investigated by the ultrastructural cytochemical techniue and by biochemical assay on homogenates of eggs from before fertilization to the 2-cell stage. Biochemical assays showed that the unfertilized eggs had strong catalase activity whereas fertilized eggs had weak activity owing to the rapid decrease of activity after fertilization. The activity did not change from immediately after fertilization to the 2-cell stage. Cytochemical examination showed that the peroxidatic activity of catalase was mainly localized in the lamellae in the cortical granules. Disintegrated cortical granules with no lamellae and substances in the perivitelline space derived from breakdown of the cortical granules had no peroxidatic activity of catalase.