Isotopic Labeling of Embryo, Yolk Sac, and Intracellular Parasites of the Embryonated Hen''s Egg by Yolk Replacement

A technique is described which allows the replacement of 50% of the yolk of the embryonated hen's egg with large volumes of diverse but chemically defined solutions. By using an electrosurgical unit and a polyethylene tunnel, the procedure was performed on eggs from days 3 through 7 with greater than 90% surgical success and viability for the short term. More than 50% of the eggs replaced showed viability for 2 weeks, and a significant proportion went full term. ³²PO₄ and amino acids (³H and ¹⁴C) added to the replaced eggs were incorporated into the macromolecules of the embryo and yolk sac as well as into parasitic rickettsiae cultivated in the replaced eggs. The incorporated ³²PO₄ was shown to be assimilated into a variety of biochemical species.     

Initiation of Yolk Deposition by Juvenile Hormone

JUVENILE hormone is secreted by the corpus allatum gland, in insects. It was first implicated in the control of yolk deposition in 1936¹ and has recently been found to stimulate fat body synthesis of vitellogenic blood proteins (vitellogenins)^2,3 which are selectively incorporated into yolk by the oocytes^4–6.     

Evidence from triton X-100 polyacrylamide gel electrophoresis that histone f2a2, not f2b, is phosphorylated in Chinese hamster cells

Chinese hamster cells (line CHO) were labeled in suspension culture with ³H-lysine and ³²PH₃PO₄. Preparative polyacrylamide gel electrophoresis of histone fractions from these cells was performed in the presence of 8 $\text{M}$ urea, 6 mM Triton X-100, and 0.9 N acetic acid. This method separates histones f2a2 and f2b by a Large distance, thus making it possible to resolve the controversy concerning which histone -- f2b or f2a2 -- is phosphorylated. It is shown that the two most highly phosphorylated histones in interphase CHO cells are f1 and f2a2. Histones f2b and f3 are shown to contain no significant incorporation of ³²PO₄ in interphase cells, while histone f2a1 contains a small but detectable amount of incorporated ³²PO₄. Binding of the nonionic detergent Triton X-100 to hydrophobic centers appears to be greatest for histones f2a2 and f3, thus significantly retarding the mobility of these two histones during electrophoresis.     

AMMONIUM,NITRATE, PHOSPHATE,AND INORGANIC CARBON UPTAKE IN AN OLIGOTROPHIC LAKE: SEASONAL VARIATIONS AMONG LIGHT RESPONSE VARIABLES1

The dependence of substrate saturated uptake of ¹⁵NH₄⁺, ¹⁵NO₃^?, ³²PO₄^3?, and ¹⁴CO₂ on photosynthetic photon flux density (PPFD or photsynthetically active radiation, 400–700 nm) was characterized seasonally in oligotrophic Flathead Lake, Montana. PO₄^3? uptake was not dependent upon PPFD at any time of the year, whereas NH₄⁺, NO₃^?, and CO₂ uptake were consistently dependent on PPFD over all seasons. Maximal rates of NH₄⁺, NO₃^? and CO₂ uptake usually occurred near 40% of surface PPFD, which corresponded to about 5 m in the lake; inhibition was evident at PPFD levels greater than 40%. NH₄⁺, NO₃^? and PO₄^3? were incorporated in the dark at measurable rates most of the year, whereas dark CO₂ uptake was always near 0 relative to light uptake. CO₂ and NO₃^? uptake were more strongly influenced by PPFD than was NH₄^3? uptake. The PPFD dependence of PO₄^3?, NH₄⁺, NO₃^? and CO₂ uptake may affect algal growth and nutrient status by influencing the balance in diel and seasonal C:N:P uptake ratios.     

Phosphorus-32, a Clinically Available Drug,Inhibits Cancer Growth by Inducing DNA Double-Strand Breakage

Radioisotopes that emit electrons (beta particles), such as radioiodine, can effectively kill target cells, including cancer cells. Aqueous ³²P[PO₄] is a pure beta-emitter that has been used for several decades to treat non-malignant human myeloproliferative diseases. ³²P[PO₄] was directly compared to a more powerful pure beta-emitter, the clinically important ⁹⁰Y isotope. In vitro, ³²P[PO₄] was more effective at killing cells than was the more powerful isotope ⁹⁰Y (P ≤ 0.001) and also caused substantially more double-stranded DNA breaks than did ⁹⁰Y. In vivo, a single low-dose intravenous dose of aqueous elemental ³²P significantly inhibited tumor growth in the syngeneic murine cancer model (P ≤ 0.001). This effect is exerted by direct incorporation into nascent DNA chains, resulting in double-stranded breakage, a unique mechanism not duplicatable by other, more powerful electron-emitting radioisotopes. ³²P[PO₄] should be considered for human clinical trials as a potential novel anti-cancer drug.     

The synthesis of phosvitin in vitro by slices of liver from the laying hen

1. Slices of liver from laying hens incorporated Na₂¹⁴CO₃ and NaH₂³²PO₄ into phosvitin. Slices of liver from immature birds did not do so to any appreciable extent. The ³²P was incorporated into O-phosphorylserine in the phosvitin molecule. 2. Kidney, spleen, muscle, large and small intestine, ovary and oviduct from laying birds did not incorporate Na₂¹⁴CO₃ into phosvitin. 3. Slices of liver from laying hens carried out a net synthesis of phosphoprotein under the standard conditions of incubation. Slices from the livers of immature pullets did not do so. 4. Liver from the laying hen incorporated [2-¹⁴C]glycine, [3-¹⁴C]serine and [2-¹⁴C]glutamic acid into phosvitin. Part of the glycine was shown to be present as serine in the final product. 5. Slices of liver from immature birds treated with oestradiol synthesized phosvitin from [2-¹⁴C]glycine, but the addition of oestrogens in vitro to slices from untreated immature birds did not promote synthesis during a 3 hr. incubation period.     

Role of silicon in diatom metabolism

Proteins of Cylindrotheca fusiformis which incorporated significant ³²PO₄ were identified as soluble, acidic proteins, and their two-dimensions gel positions were determined. Upon addition of silicate to silicon-starved cells, at least 3 of these proteins showed a significant and rapid change in the level of phosphorylation. Under the same conditions the amount of ³²PO₄-labeled ATP, ADP, and GTP remained relatively constant. Thus silicon appears to affect phosphorylation and dephosphorylation of specific proteins, and these changes are sufficiently rapid to suggest that phosphorylation may have a role in mediating the silicon requirement for both DNA synthesis and the accumulation of specific mRNAs.     

Evidence for selective regulation of the phosphorylation of myocyte proteins by isoproterenol and prostaglandin E1

Both isoproterenol and prostaglandin E₁ increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with ³²PO₄^3?, 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated ³²PO₄^3? incorporated into 16 proteins, including 2 phosphoproteins not observed under control conditions. By contrast, prostaglandin E₁ neither caused a measurable change in the protein phosphorylation pattern nor interfered with isoproterenol's capacity to do so. Isoproterenol stimulated myocyte protein phosphorylation in either the presence or absence of extracellular Ca²⁺. The results suggest that the regulation of protein phosphorylation following adenylate cyclase stimulation is: (1) an agonist-specific process and not due solely to a random accumulation of intracellular cycle AMP and activation of protein kinase; (2) the Ca²⁺ mobilization component of β-receptor activation does not account for the paradoxical effects of isoproterenol and prostaglandins E₁; (3) activation of cyclic AMP-dependent protein kinase does not always result in an enhancement of protein phosphorylation.     

Water quality of the Modder River,South Africa

The Modder River is a relatively small river in the central region of the Free State Province, South Africa and has a mean annual runoff of 184 × 106m³. Botshabelo is a city, which has developed in the catchment area of the river, and its sewage outflows are discharged into the Klein Modder, a tributary of the Modder River. This study was conducted in order to determine the influence of Botshabelo's sewage outflow on the water quality of the river. It was determined that the Modder and Klein Modder Rivers do not generally follow distinctive seasonal patterns in terms of chemical parameters, although NO₃-N and PO₄-P concentrations usually increase with increasing flow and conductivity decreases with increasing flow. Physical parameters such as turbidity, flow and temperature did however follow distinctive seasonal patterns from February 1996 to December 1997, as did phytoplankton growth. Low chlorophyll-a concentrations were exhibited in the winter and higher concentrations during spring. In the Klein Modder River, algal blooms occurred more frequently, and the algal biomass was higher than in the Modder River. This could be ascribed to the higher nutrient concentrations and lower flow velocities in the former. The inflow of the Klein Modder River into the Modder River caused on average, 112% increase in PO₄-P, 171% increase in NO₃-N, 50% increase in chlorophyll-a concentration, and 230% increase in E. coli counts.     

Radio-manganese, -iron,and -phosphorus uptake by water hyacinth and economic implications

The proliferation of an aquatic plant, water hyacinth,Eichhornia crassipes, has prompted investigation into the nutrient requirements of this plant, as well as its capacity for removing micronutrients (selected transition metal ions and phosphorus) from fresh water. The apparent stimulation of growth by iron and manganese led to experiments to evaluate the rate of uptake of these two elements, present as the EDTA salts, in comparison with phosphorus. One-compartment experiments indicate all three elements are actively absorbed by the root systems, but the rates of absorption and translocation differ markedly. Specifically, phosphorus as³²PO₄ is taken up by the roots and appears in the plant leaves within 48 hours, followed by a slow but constant phase of uptake of³²PO₄. In contrast, iron, as⁵⁹Fe(III), is rapidly taken up by the roots and only at 21 days do experimentally significant quantities of the radio-element appear in the leaves. Manganese-54 enters the roots as rapidly as iron, but the rate of appearance of manganese in the leaves is about ten times faster. The implications in terms of removal of micronutrients are considered.     

Nuclear incorporation of P32 as demonstrated by autoradiographs

Roots of Vicia faba seedlings were grown for periods of 2 to 48 hours in solutions of P³² in the form of NaH₂PO₄. Stripping-film autoradiographs were made of squashes and sectioned material from the meristem and from proximal segments where cell divison is rare. It is concluded (a) that P³² enters into organically bound form (probably desoxyribonucleic acid) in the nucleus during the resting stage, but not during the actual division of the cell; (b) that P³² is incorporated in this form only in nuclei which are preparing for division; (c) that P³² in this form is inherited by the daughter cells.     

Yolk organelles and their membranes during vitellogenesis ofXenopus oocytes

Summary The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Throughout vitellogenesis, large yolk platelets are in close contact with smaller nascent yolk organelles. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets was not observed. On freezeetching replicas, yolk-platelet membranes present fracture faces with intramembranous particles (IMP) of various sizes and a heterogeneous distribution of approximately 200–600 IMP/μm² at the E face, and 1200–2100 IMP/μm² at the P face. Again, this presentation of the membrane exhibits neither anastomoses to the endoplasmic reticulum, nor caveolae that exclude the uptake of yolk-containing vesicles into these yolk organelles. Proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.     

Composition,secretion kinetics and radioactive labelling of the extracellular slime polysaccharide secreted during spherulation ofPhysarum polycephalum

The myxomycetePhysarum polycephalum synthesizes copious amounts of slime when differentiating into the hard walled resting stage. The chemical composition of slime obtained after introduction of spherulation in a nutrient and a non-nutrient salt-medium has been analysed and compared. The composition of slime is almost identical after the two different induction methods. This slime could be labelled with radioactived-[U-¹⁴C]glucose,³²PO₄ ^3–,³⁵SO₄ ^2– and⁷⁵SeO₄ ^2–. The kinetics of slime secretion after both induction methods has been followed using different criteria. The sulfate analog⁷⁵SeO₄ ^2– seems to be incorporated into the slime, partially replacing sulfate groups of the sulfated polysaccharide. Furthermore,d-[U-¹⁴C]glucose was used to labe the spherule walls. Determination of an alkali resistent polysaccharide component serves as a new method to follow wall formation.     

Influence of phytoplankton on the response of bacterioplankton growth to nutrient enrichment

1. Laboratory experiments were conducted to test the effect of nutrient enrichment on bacterioplankton growth in the presence and absence of phytoplankton. 2. In one series of experiments, bacterioplankton growth in terms of specific activity [³H-thymidine incorporation (cell number)^?1] was greater in whole lake water samples than in samples from which phytoplankton had been removed by filtration (1.0 μm), regardless of the nutrient enrichments (control, NH⁺₄ plus PO^3-₄ and mannitol). Organic C enhanced bacterioplankton growth in both whole and filtered lake water. 3. In another series of experiments (with the same nutrient enrichments as in the first experiment except that glucose replaced mannitol), bacterioplankton growth in whole lake water enriched with PO^3-₄ plus NH⁺₄ and incubated in the light was greater than in two treatments designed to inhibit photosynthetic activity (+DCMU and dark). Bacterioplankton response to nutrient addition was greatest in the PO^3-₄ plus NH⁺₄ enrichment under all three conditions (light +DCMU, and dark). 4. These results indicate that bacterioplankton growth could be directly limited by inorganic P and N when these elements are in short supply. Enhancement of bacterioplankton growth by phytoplankton occurs only under PO^3-₄ and NH⁺₄ replete environments.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Proteolytic processing of Blattella germanica vitellin during early embryo development

In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization is initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt's processing, its specific activity with synthetic substrates increased four-fold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt's large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (B) processed the M_r 102,000 subunit but not the M_r 95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant's eggs at day 6 mirrored the extent of processing, being highest in group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It is concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic examination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diameters of 15–30 μm to 3–10 μm. Yolk granules of those translocant's eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally.     

Yolk polypeptide gene expression in Minute Drosophila females

Minutes have been considered for some time to be mutant at the sites of synthesis of some components of the protein synthetic apparatus. To study the hypothetical relationship between Minutes and suboptimal translation, a group of abundant proteins, the yolk polypeptides, was assayed in outcrossed females bearing M(3)w, M(3)h ^y , or M(1)n mutations. Recently emerged Minute females contained a lower amount of yolk polypeptides, in both ovarian and nonovarian tissues, than their non-Minute sisters. This low level correlated with the lower abundance of cytoplasmic RNA in Minutes compared to control females. By 1 week of age, both M(3)w and their non-Minute sibs contained the same amount of yolk polypeptides and the corresponding mRNA. The double heterozygote, ap ⁴/+;M(3)w/+, did not differ in yolk polypeptide content from control flies. M(3)w females demonstrated reduced fecundity during the period of low yolk polypeptide content but gradually increased egg deposition as yolk polypeptide levels rose. These results suggest that the low protein levels are due to the slower maturation of M(3)w, and not to less efficient translation machinery.This work was supported by the NSERC (Canada) and a Queen's University ARC grant.     

Demonstration of the formation of 1,3-diphosphoglyceric acid as an intermediate in the high-energy phosphate metabolism during reinitiation of development in Artemia

Extensive labelling of the glycolytic intermediate 2,3-diphosphoglycerate by ³²PO^3?₄ during the early periods of development in Artemia is reported. At 30 min of activation this is the major labelled compound. The mobilization of inorganic phosphate through glycolysis leading to the formation of 1,3-diphosphoglycerate results in the formation of a high-energy phosphate donor. The label from this compound could be chased to high-energy phosphates (adenine derivatives). The location and subsequent high degree of labelling of 2,3-diphosphoglycerate in the yolk platelets further demonstrate the important role played by this organelle in the metabolic events accompanying the breakdown of dormancy in Artemia.     

The action of overdosed biotin on reproduction of the hide beetle,Dermestes maculatus

Excessive biotin (cis-tetrahydro-2-oxothieno[3,4-d]-imidazoline-4-valeric acid) intake by female Dermestes maculatus permits protein incorporation into yolk but suppresses embryogenesis during its later stage, presumably due to partial inactivation of egg proteases. Experiments with dietary biotin-carbonyl-¹⁴C suggested that the overdosed vitamin forms a complex with insoluble yolk proteins. The superfluous vitamin does not curtail the activity of acid phosphatase in young embryos. Hide beetles, sterilized by 1·0% dietary biotin, incorporate to their eggs about 2·3 times more biotin than control females, whereas the former excrete about 27·4 times more biotin than the latter. The adults seem to eliminate the vitamin surplus less efficiently than the larvae. The significance of impaired utilization of yolk proteins is discussed as a means for insect chemosterilization.     

Distribution of incorporated and synthesized protein among cell fractions of Xenopus oocytes

Developing oocytes of Xenopus laevis were isolated, pulsed for 10 minutes with either vitellogenin-³H, ³²P or a mixture of l-leucine-³H and ³²P_i, and subsequently incubated for various lengths of time in unlabeled medium. Homogenates were then prepared and centrifuged on 20–60% sucrose gradients.Vitellogenin-³H, ³²P was found to associate initially with membranous material, but within 45 min more than half the label was associated with the yolk platelets. Since it takes at least 60–120 min for vitellogenin to be converted into lipovitellin and phosvitin, this transformation must occur within the platelets rather than at the oocyte surface or within pinosomes.Eighteen hours after a pulse with l-leucine-³H and ³²P_i, neither the yolk nor the mitochondria fraction became significantly labeled; this indicates that the macromolecular components of these structures are not synthesized or phosphorylated by the oocyte to any great extent during vitellogenesis. Instead, various components within the membranous and “soluble” regions became labeled.