On the Mode of Activation of Sequestered Messengers in Artemia salina

Activation of the dormant embryos of Artemia salina was marked by a rapid increase in ³²P uptake which reached a stationary phase after 6 h of activation. The increase in ³²P uptake by whole cysts was paralleled by its incorporation into nucleotides. Fractionation of acid-soluble nucleotides and alkaline hydrolysate of nucleic acids on Dowex-1-formate column revealed the ³²P radioactivity to be exclusively localised in AMP. Analysis of the labelled RNA species extracted at different stages of development indicated a preferential labelling of small molecular weight species till the emergence of the embryos, followed by the de novo synthesis of messenger and stable RNA species in later stages of development. During early development, polyadenylated RNA species were localised in the par-ticulate fraction sedimenting at 16,000 rpm and their location shifted to the soluble fraction as development proceeded. Activation of preformed messengers by phosphorylation of the adenylate residue of their poly A stretches and translocation of the capacitated messengers to the cytosol via a RNP-membrane complex is proposed as a trigger of embryonic differentiation.     

Studies on Polyadenylic Acid-Containing RNA from Developing Nervous System of the Chicken the

Abstract: To investigate certain biochemical aspects of myelination, a study was undertaken of the messenger-like RNA in the nervous system of pre- myelinating 14-day embryos and of myelinating 17-day embryos and 3-day chicks. The central and peripheral nervous systems of the chick were found to contain and to actively synthesize poly(A)⁺ RNA. RNA species binding to oligo(dT)-cellulose contained a relatively high proportion of adenylate residues and were resistant to the actions of pancreatic and T₁ ribonucleases. Preparations labeled by incubation with adenosine in vitro showed a decrease in the proportion of poly(A)⁺ RNA as the age of the animal increased, while preparations labeled in vivo exhibited the opposite trend. Polyacrylamide gel electrophoretograms of both in vivo and in vitro labeled pqeparations showed that the poly(A)⁺ fractions contained mainly heterodisperse RNA species. The average molecular size of poly(A)⁺ RNAs of purified polysomal fractions of nerve RNA from 3-day chicks was smaller than 18S, whereas that of total poly(A)⁻ RNA was larger than 18s. The proportion of poly(A)⁺ molecules larger than 18s was lower in the rapidly myelinating nerve tissues of 17-day embryos and post-hatching chicks than in those of premyelinating 14-day embryos. Similar results were obtained for crude nuclear RNA fractions or RNA preparations fractionated under denaturing conditions. These results are consistent with previous work showing that the embryonic peripheral nerve contains a larger proportion of high-molecular-weight, messenger-like RNA molecules than does nerve tissue from young chicks or adults.     

Accumulation and Spatial Distribution of Poly(A)+RNA in Oocytes and Early Embryos of Drosophila melanogaster

We describe the accumulation and distribution of poly (A)⁺RNA during oogenesis and early embryogenesis as revealed by in situ hybridization with a radio-labeled poly (U) probe. The amount of poly (A)⁺RNA in nurse cell cytoplasm continuously increased untill mid-vitellogenic stage (st. 10), then decreased with the rapid increase of poly (A)⁺RNA in the oocyte (st. 11). The localization of poly (A)⁺RNA at stage 10 was in the anterior region of the oocyte, where it is connected by cytoplasmic bridge to the nurse cells. These observations indicate that most of the poly (A)⁺RNA synthesized in the nurse cells is transferred to the oocyte through the cytoplasmic bridges at stage 10–11. During the remainder of oogenesis (st. 11–14) and during preblastodermal embryogenesis, poly (A)⁺RNA was evenly distributed over the cytoplasm of oocytes and embryos. At blastoderm stage, poly(A)⁺RNA became concentrated in the peripheral region of embryos. Though the somatic nuclei of the blastoderm contained a detectable amount of poly (A)⁺ RNA, the pole cell nuclei did not. The cytoplasmic RNA visualised by acridine orange staining and the poly (A)⁺RNA detected by hybridization with [³H]poly (U) exhibited identical distributions during oogenesis and early embryogenesis. These observations provide a basis to assess the unique distributions of specific RNA sequences involved in early development.     

Renewal of sexual receptivity in mated female mosquitoes, Aedes aegypti

ABSTRACT. Females of Aedes aegypti (L.) (Diptera, Culicidae) were not receptive to mating 1 h after the initial insemination or at later times throughout the first gonotrophic cycle. A significant proportion of these females (75–90%) were inseminated again prior to the second gonotrophic cycle. Insemination was determined by the detection in females of sperm labelled with ³ H(G)-adenine or ³²PO₄ after matings with radiolabeled males. Females deprived of males after the first insemination displayed an abnormal reduction in fertility throughout successive gonotrophic cycles.     

LOCALIZATION AND SEGREGATION OF MATERNAL RNA'S DURING EARLY CLEAVAGE OF XENOPUS LAEVIS EMBRYOS

The localization and segregation of maternal RNA's during early cleavage of Xenopus laevis embryos were studied. Blastomeres and hemispheres of eggs and early embryos were separated manually and the amounts of ribosomal RNA and poly(A) ⁺RNA extracted from each blastomere and hemisphere were determined by optical density measurement and by ³H-poly(U) hybridization, respectively. It was found that both kinds of the maternal RNA's were more abundant (two-thirds of the total) in the animal hemisphere (cells), while they were evenly distributed between the dorsal and ventral halves. This pattern of localization remained unchanged from the egg to the blastula stage, indicating that these maternal RNA's were segregated into blastomeres quite simply by cell division. Gel electrophoresis showed that the size distributions of poly(A) ⁺RNA and poly(A) sequences obtained from different blastomeres of 8-cell embryos did not differ greatly. It was also found that cytoplasmic polyadenylation of maternal RNA, which occurs during early cleavage and blastulation, took place equally in all regions of the cleaving embryos, suggesting no regional difference in the localization of maternally inherited nonpolyadenylated RNA. These observations are discussed in relation to previous findings on differences along the animal-vegetal and dorsal-ventral axes of the early amphibian embryo.     

Alterations of Lipid Metabolism in Response to Nerve Growth Factor

Abstract: In response to nerve growth factor (NGF), clonal pheochromocytoma cells flatten and extend neurites capable of making functional synapses. Although no significant changes in overall phospholipid composition occur in the presence of NGF, there is increased incorporation of ³²PO₄ into phosphatidylinositol and phosphatidic acid within 10 min after the addition of NGF. NGF stimulates the incorporation of ³²PO₄ into other lipids, such as phosphatidylcholine, to a lesser extent. The kinetics of the NGF-induced phosphatidylinositol responses are different when the cells are in suspension from when they are attached to culture dishes. These changes in phospholipid metabolism are discussed with respect to their role in NGF-induced nerve differentiation.     

STUDIES ON THE POLYSACCHARIDE-SULFATING SYSTEM IN SEA URCHIN EMBRYOS

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [³⁵S]sulfate in vivo and [³⁵S]3'-phosphoadenosine 5'-phosphosulfate ([³⁵S]PAPS) in a cell-free system. In vivo incorporation of [³⁵pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [³⁵S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [³⁵pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg ²⁺and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

GENERAL PATTERN, 35SO4-INCORPORATION AND INTRACELLULAR LOCALIZATION OF GLYCANS IN DEVELOPING SEA URCHIN (ANTHOCIDARIS) EMBRYOS

Glycosaminoglycan (GAG) prepared from sea urchin embryos ( Anthocidaris crassispina ) at various stages with or without pulse ³⁵SO₄-labelling was separated into various fractions by chromatography on DEAE-cellulose with a linear NaCl concentration gradient: fraction "P" (nonacidic) and fractions "A" through "F" (of increasing acidities). The ³⁵SO₄-radioactivity was negligible in "P" and "A", largest in "B" and "C", and decreased in the other fractions three alphabetical order. During development (hatched blastulae to gastrulae) the glycans in fractions "P" and "A" decreased in amount, whereas those in "E" and "F" increased. "E" contained heparin-like (AMPS-1) and dermatanpolysulfate-like (AMPS-2) GAG in addition to a sulfated fucogalactan-like (E₁) glycan. Another sulfated fucogalactan-like (F₁) glycan was found in "F". A sulfated polysialic acid-like (S₁) glycan was found in "C". An EDTA-extract of gastrulae gave AMPS-2, E₁ and F₁. The mitochondria-rich fraction gave AMPS-1, whereas the yolk granule-rich fraction gave S₁. Most of the other still unidentified components in "B", "C", and "D" appeared to be derived from glycoproteins and were mainly located in the crude yolk-mitochondrial and cytosol fractions.     

Phosphorus transformations in the epilimnion of humic lakes: abiotic interactions between dissolved humic materials and phosphate

SUMMARY. 1. Sephadex gel filtration of filtered water from small, Finnish forest lakes demonstrated abiotic movement of ³³P from added PO₄ to two higher molecular weight fractions. This movement was most pronounced in waters with high humic content which also had high iron content. The two fractions which took up ¹³P had nominal molecular weights of > 100,000 and 10,000-20,000.
2. An equilibrium existed between free PO₄ and the two fractions. However, one fraction, at least, appeared to exist in two phases, with one phase in rapid equilibrium with free PO₄ but the other in only slow equilibrium.
3. Additions of ferric iron up to 1 mg Fe l⁻¹ to the filtered lake water stimulated movement from free PO₄, provided high concentrations of humic materials were present. In the absence of humic materials even 0.1 mg Fe 1⁻¹ would precipitate all added ³³PO₄.
4. The high molecular weight P was only partially reactive with standard molybdate reagents. Exposure of the high molecular weight P to sunlight caused a small release of PO₄ under the experimental conditions employed.
5. Possible implications for biological phosphorus demand of such sequestration of free PO₄ by humic materials in combination with iron are discussed.     

Measurements of phosphorus uptake by macrophytes and epiphytes from the LaPlatte River (VT) using 32P in stream microcosms

1. Phosphorus (P) uptake by macrophytes and epiphytes from the LaPlatte River (VT) was examined in the laboratory by adding ³²PO₄‐P to recirculating stream microcosms.
2. Water, plugs of sediment and plants were removed from the river and placed into the microcosms. ³²PO₄‐P was then added either to the water or the sediment, and its incorporation into plants and epiphytes was monitored over 3 days. Uptake was examined at both ambient (5 μg L^–1) and increased (50 μg L^–1) soluble reactive phosphorus (SRP) concentrations. A computer program was developed to fit curves to the radiotracer data and calculate rate constants for the simultaneous transfer of ³²P among compartments.
3. Both macrophytes and epiphytes removed P from the water, but epiphyte uptake of P was more rapid. Phosphate enrichment stimulated P uptake by both macrophytes and epiphytes. Macrophytes also obtained P from the sediment. The relative contribution of P to macrophytes from the water vs. that from the sediment appeared to vary with SRP in the overlying water. Accurate estimates of rates of P uptake from sediments by macrophytes were difficult to obtain however, due to very low and highly variable unit rate constants for P uptake and uncertainty about the magnitude of the phosphate pool available for uptake.
4. SRP concentrations were greater in the overlying water than in the sediment pore water of stream microcosms in the present study. Numerous reports in the literature have suggested that this condition favours uptake by macrophyte stems and leaves rather than by roots.
5. Phosphate uptake from the water by macrophytes in shallow streams may be more common than for macrophytes in lakes.     

Agonist-Induced Endocytosis of Muscarinic Cholinergic Receptors: Relationship to Stimulated Phosphoinositide Turnover

Abstract: The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and α subunits of G_q and G₁₁, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V₁). Agonist-induced increases in diacylglycerol mass and in ³²P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V₁ fraction, but these increases persisted when the agonist-induced translocation of receptors into the V₁ fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V₁ fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both ³²P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.     

Defects in the archenteron of mouse embryos homozygous for the T–mutation

Abstract. The development of the head process was studied in mouse embryos homozygous for the Brachyury (7) mutation at stages between days 7¹/₂ and 8¹/₂ using scanning electron microscopy. Intact T/T embryos removed from deciduae were distinguished from their normal littermates at the presomite stage on the basis of an abnormally short allantois. In the homozygotes so distinguished, the prenotochordal cells were fewer in the archenteron area, and the typical indentation of the archenteron was not observed. These defects are probably early indicators of the abnormality of the notochord previously described for later stages of development.     

RNA and protein metabolism during adventitious root formation in stem cuttings of Phaseolus aureus

Indole-3-butyric acid (IBA, 10⁻⁴ M ), spermine (7 × 10⁻⁵ M ) and vitamin D₂ (6.3 × 10⁻⁵ M ), all of which enhance rooting in mung bean cuttings ( Phaseolus aureus Roxb. cv. Berkin), influence RNA metabolism. Total and poly (A)⁺-RNA synthesis within the hypocotyl is inhibited by each of these chemicals within 24 h. These changes precede induced cell division and are therefore associated with the so-called inductive period of regeneration during which some cells in the hypocotyl undergo dedifferentiation. However, following subsequent transfer of cuttings to borate, which is an essential prerequisite for development of root primordia in these cuttings, RNA synthesis is enhanced by pretreatments with IBA, spermine or vitamin D₂. Furthermore, IBA inhibits synthesis and turnover of protein within the hypocotyl.     

Influence of aluminium on phosphorus and calcium localization in roots of beech (Fagus sylvatica)

Beech plants ( Fagus sylvatica L. provenance Maramures) were grown in nutrient solution at low pH (4.2) and exposed to different concentrations of AlCl₃. Uptake and leakage of Ca²⁺(⁴⁵Ca²⁺) and H²PO₄-(³²P) were studied. A high external aluminium concentration (1.0m M ) reduced the uptake and export to the shoot of both calcium and phosphate, while 0.1 m M Al increased the phosphorus level in the roots. To determine the impact of aluminium on the localization of calcium and phosphate, leakage of the elements from both intact plants and plants frozen prior to the leakage experiment was studied. The leakage of Ca²⁺ from intact plants was not affected by prior exposure to 0.1 m M Al. Freezing of the beech plants before the leakage experiment increased leakage of calcium slightly more from roots of control plants than for roots exposed to 0.1 m M Al, indicating that even low concentrations of alminium may impede the influx of calcium across the plasma membrane in the roots. The patterns of Ca²⁺ leakage from roots previously exposed to 1.0 m M Al indicated that very little Ca²⁺ was located extracellularly. The extracellular fraction of phosphate increased with increasing Al concentration in the nutrient solution. Low Al concentration (0.1 m M ) only reduced the intracellular phosphate concentration to a minor extent, while 1.0 m M Al profoundly decreased it. It is concluded that 0.1 m M AlCl₃ has a limited effect upon the localization of Ca²⁺ and phosphate in the roots. At higher levels of Al, 0.1–1.0 m M , there is a more dramatic change in nutrient localization in the free space and uptake over the plasma membrane.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis

Abstract The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis . Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD₅₀ of 10³ colony forming units (CFU) or greater (low virulence) from those having an LD₅₀ of approximately 10¹ or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica , a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD₅₀ of 10² were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.