Periodicity of Cytoplasmic Cycle in Nonnucleate Fragments of Sea Urchin and Starfish Eggs*

Fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus and the starfish, Asterina pectinifera were separated by manual bisection to obtain pairs of nucleate and non-nucleate fragments. Simultaneous observation on the pair revealed that the cyclic change in the cortical tension in the non-nucleate fragment was definitely prolonged than the cleavage interval of nucleate partner by about 30%. Activated non-nucleate cytoplasmic fragments derived from the unfertilized eggs and colchicine treated whole eggs of H. pulcherrimus still showed the same degree of prolongation.     

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography.
Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave.
Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.     

Cyclic Changes in Shape of A Non-Nucleate Egg Fragment of Tubifex (Annelida, Oligochaeta)

A non-nucleate fragment separated from the fertilized Tubifex egg at metaphase of the second meiosis showed temporary surface deformation at 3–3.5 hr intervals, i.e. , synchronously with the onset of formation of the second polar body and early cleavages in control eggs. From the two-cell stage on, the periodicity of the surface activity in the non-nucleate fragment was found to be synchronous with the cleavage cycles of the CD-cell and its descendants, but not with those of the AB-cell. This surface deformation was completely inhibited by cytochalasin B (50 μg/ml). Electron microscopy shows that microfilaments are present exclusively in the cortical layer of the deforming fragments. Cycloheximide-treated egg fragments commenced surface deformation after a delay of 1–2 hrs; pulse-treatment indicated that the surface deformation requires proteins synthesized specifically during the period of the previous surface deformation. These results are discussed in relation to the nucleus-independent cytoplasmic rhythm and asynchronous cleavage of Tubifex eggs.     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

Histone acetylation during meiosis in Lilium microsporocytes

Acetate incorporation into histones of Lilium microsporocytes is consistently higher in early prophase of meiosis than in later stages. The pattern of acetate incorporation into histones differs from that found for the soluble nuclear proteins. Evidence is presented that acetate incorporation reflects true acetylation, not histone synthesis. e-N-acetyllysine (eNAcLys) was released from histones by enzymatic digestion. Amino acid analysis of the digests confirm the presence of significantly higher amounts of eNAcLys in early meiotic stages. These results are consistent with the hypothesis that modulation of histone charge plays a role in the binding of histones to DNA and/or chromosome condensation. In vitro levels of histone acetylating activity remain constant throughout meiosis. The results suggest that microsporocyte histone deacetylase activity increases through meiotic prophase.     

Inhibition of oleic acid incorporation into a non-lipid fraction by chloroacetamide herbicides

Oleic acid is incorporated into an insoluble fraction left over after lipid extraction in Scenedesmus acutus. This incorporation is extremely sensitive to the chloroacetamide herbicide, metazachlor (I₅₀= ca 20 nM). Therefore, factors influencing the incorporation of radioactivity from oleic acid into this non-lipid fraction were investigated. S. acutus cells were cultivated under various conditions with or without inhibitors and [¹⁴C]-oleic acid was supplied to the algae; the lipids were extracted and the radioactivity incorporated in the remaining fraction monitored. The inhibition seemed specific for chloroacetamides and related classes since it was also observed with alachlor, dimethenamid and mefenacet (an oxyacetamide). In contrast, it could not be found with diuron, oryzalin, nor could it be observed with a non-herbicidal metazachlor derivative or iodoacetamide. Incorporation of oleic acid into that fraction required meta-bolically active cells and was stimulated by light. Other fatty acids (16:0, 18:2, and 18:3) were also incorporated into the non-lipid fraction but their incorporation was not inhibited by metazachlor. Among other components, the fraction contains proteins. However, a possible specific effect of chloroacetamides on the binding of oleic acid to proteins or on the in vitro activity of lipid transfer proteins could not be detected. Not much is known yet about mechanism and chemistry of oleic acid incorporation but this finding opens a new path for investigations towards the primary target of these herbicides.     

Mitochondrial regulation in sea urchins. III. Rapid degradation of mitochondrial RNA in association with a failure to form mitochondrial polyribosomes in eggs activated with ionophore A 23187.

Most of the biochemical changes which have been examined in eggs of sea urchins, following activation with ionophore A-23187, parallel those events which have been observed to occur after fertilization. However, the results reported here indicate that mitochondrial polyribosomes fail to form after ionophore activation of either nucleate or anucleate fragments of eggs of the sea urchin. The results also demonstrate that ionophore activation does not impair the ability of eggs to generate ATP within the subsequent 2.5 h and thus the absence of mitochondrial protein synthesis appears not to be responsible for the failure of ionophore activated eggs to divide. Studies of the rates of uptake and incorporation of [³H]uridine into nucleic acid within ionophore activated anucleate fragments suggest that the rates of synthesis and degradation for both messenger and ribosomal “like” RNAs reach equilibrium within 5 min after the addition of isotope to the cultures, demonstrating that the half-lives of newly synthesized mitochondrial RNAs may be relatively brief in the absence of polyribosome formation. These results support and extend the conclusion of Lambowitz et al. [42] which suggests that ribosomal proteins may be important for stabilization of at least one of the mitochondrial ribosomal RNAs.     

Die Wirkung von Rifampicin auf die RNA- und Proteinsynthese sowie die Morphogenese und den Chlorophyllgehalt kernhaltiger und kernloser Acetabularia-Zellen

Summary Rifampicin (10g/ml) strongly inhibits the incorporation of [5-³H]-uridine into the chloroplast RNA of anucleate cells of the green alga Acetabularia mediterranea, whereas incorporation into nuclear RNA is hardly affected.Furthermore, at a concentration range of 1–10g/ml rifampicin has only a small effect on stalk- and cap formation in nucleate posterior parts of the stalk. As has already been shown, the morphogenesis of such cell segments depends on the synthesis of new RNA in the nucleus. Similarly rifampicin only slightly inhibits the synthesis of the enzyme UDPG-pyrophosphorylase, which is coded by nuclear DNA.These slight inhibitions are interpreted as secondary effects arising from a blockage of plastid RNA synthesis, since both nucleate and anucleate cells respond in a similar manner and to the same degree.In contrast the increase in the chlorophyll content in nucleate and anucleate cells is severely impaired by the antibiotic. These findings indicate that the nucleus and the plastids contain different DNA-dependent RNA-polymerases.     

Activation of protein kinase(s) by glucagon and cyclic AMP in the rat liver: Relationship to metabolic effects

Although it is known that protein kinases are activated by cyclic AMP, the role of the activated kinase in the gluconeogenic response to cyclic AMP is not known. Therefore, we examined whether the inhibition of the gluconeogenic resposne in the liver is due to an interference with the activation of protein kinase in the following situations: (1) adrenalectomy, (2) Na⁺-free perfustae, (3) administration of local anesthetic. We measured protein kinase activity indirectly by measureing incorporation of ³²P into proteins of the perfused liver, and directly by measuring the enzyme activity. We found no significant inhibition of activation of protein kinase in teh above experimental conditions. It seems that in the intact liver, activation of protein kinase by itself is not sufficient to evoke metabolic responses.In order to clarify whether teh requirement for ion redistribution is specific for the gluconeogenic response or not, the lipolytic and antilipogenic effects of glucagon and cyclic AMP were examined. Na⁺-free persurfate, local anesta high K⁺ did interfere with the lipolytic and antilipogenic responses to these agents just as it interfered with the fluconeogenic response. It is likely that ion redistribution evoked by glucagon and cyclic AMP is essential to the expression of most, if not all, metabolic effects.     

IN VITRO SYNTHESIS OF THE MYELIN BASIC PROTEINS IN THE DEVELOPING MOUSE BRAIN: PROPERTIES OF A HOMOGENATE SYSTEM

—An in vitro system using mouse brain homogenates has been developed to study the synthesis of the myelin basic proteins. Incorporation of [³H]leucine into protein in this system did not require additional energy sources. The system was slightly stimulated by glucose and strongly inhibited by puromycin. Myelin basic proteins were isolated from incubation mixtures by conventional techniques of solvent extraction and column chromatography, and finally separated into the large and small components by polyacrylamide gel electrophoresis in an acetic acid-urea system. Gels were stained, sliced, dissolved, and counted, and relative rates of incorporation of label into the two basic proteins were determined at several ages. The ratio of radioactivity incorporated into the small (S) and large (L) basic proteins, over a 30 min incubation period, was found to increase from 0.97 at 10 days to 1.59 at 21 days and decline thereafter. These data generally agree with earlier studies on the in vivo synthesis of the myelin basic proteins in mice. An interesting feature of the time course was that incorporation of [³H]leucine into the purified myelin basic proteins relative to incorporation into total protein in the homogenate increased almost 2-fold during the course of the 30-min incubation. This suggested that post-translational processing of at least one of the two basic proteins was occurring. To examine this possibility further, experiments were conducted in which incorporation was allowed to proceed for 2–5 min, before being inhibited with puromycin; the incubation was then continued for up to 25 min longer. Although total incorporation was inhibited immediately after puromycin addition, label was found to continue to accumulate in the basic proteins to the extent of 30–100% above controls. These data support the notion that the MBPs are synthesized as precursors and then processed to yield authentic myelin basic proteins and that this processing can occur in vitro.     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α     

Diphtheria toxin inhibits the synthesis of myelin proteolipid and basic proteins by peripheral nerve in vitro

Abstract— Diphtheria toxin (DT) did not produce measurable degradation of myelin proteins or sulphatide in sciatic nerves of chick embryos after incubation in vitro for 4 h. In contrast, DT inhibited the in vitro incorporation of L-[U-¹⁴C]leucine into myelin proteins by the nerves after a delay of 1 h. Separation of the myelin proteins by SDS-polyacrylamide gel electrophoresis indicated that the synthesis of Wolfgram proteins and proteins not entering the gel was inhibited by 21–22 per cent, whereas synthesis of myelin proteolipid and basic proteins was inhibited by 79–88 per cent. Incorporation of ³⁵SO₄ into myelin [³⁵S]sulphatide was also inhibited by DT after a delay of 2 h. The inhibition of [³⁵S]sulpha-tide incorporation into myelin caused by DT differed from that observed with puromycin in that it did not depend on depletion of an intracellular transport lipoprotein. Instead, the inhibition seemed to be secondary to the decreased synthesis of myelin proteolipid and basic proteins.     

Effects of salicylic acid on protein content and <Superscript>14</Superscript>C-amino acid incorporation into proteins of pea roots

Pea (Pisum sativum L.) root treatment with salicylic acid (SA) changed the content of some proteins and incorporation of ¹⁴C-amino acids into proteins. The analysis of changes in these indices allowed us to subdivide all proteins into the four groups: (1) most abundant SA-independent proteins; (2) SA-dependent proteins, which content and ¹⁴C-amino acids incorporation both increased; (3) SA-dependent proteins, which content and ¹⁴C-amino acids incorporation both decreased; and (4) SA-dependent proteins, which content was not essentially changed (referred earlier to SA-independent proteins) but ¹⁴C-amino acids incorporation into these proteins was strongly suppressed. It is very likely that proteolysis of the proteins referred to the fourth group is very low and even a strong inhibition of their synthesis (incorporation of ¹⁴C-amino acids) does not result in the substantial decrease in their contents. Some SA-dependent proteins were identified by means of modern methods of proteomics: phosphoglyceromutase, S-adenosylmethionine synthase 3, enolase, chalcone isomerase, nucleoside diphosphate kinase 1, and tioredoxin h.     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.     

The effect of different inhibitors of transcription and translation on the expression and control of circadian rhythm in individual cells of Acetabularia

A number of inhibitors of gene expression were tested for their effect on the circadian rhythm of O₂ evolution in single nucleate and anucleate cells of Acetabularia. In the presence of actinomycin the rhythm disappeared after about 14 days in nucleate cells. Anucleate cells did not respond to the inhibitor. Rifampicin and chloramphenicol did not affect the rhythm in either nucleate or anucleate cells. Puromycin and cycloheximide inhibited the photosynthesis rhythm in both nucleate and anucleate cells. It was concluded that translation on 80 S ribosomes is essential for the manifestation of the rhythm of O₂ evolution in Acetabularia.     

Effect of Estradiol Dipropionate on Protein Synthesis in Oocytes and Ovary of the Sea Urchin Strongylocentrotus intermedius at Different Stages of the Reproductive Cycle

We studied protein synthesis in the oocytes and ovary of the sea urchin Strongylocentrotus intermedius at different stages of the reproductive cycle after treatment with estradiol dipropionate. During the early development of oocytes and active gametogenesis, this estrogen induced the incorporation of ³H-leucine in the oocytes. The changes in synthetic activity of cells were accompanied by an elevated efficient incorporation of free amino acid in proteins due to its increased pool, increased tissue permeability for precursors, and increased rate of protein synthesis. Before spawning, estradiol dipropionate did not cause any changes in protein synthesis. After estradiol dipropionate treatment, the inhibitors of protein synthesis, puromycin and actinomycin D, decreased the intensity of ³H-leucine incorporation in the oocytes and protein synthesis in the ovary. The involvement of estradiol dipropionate in the regulation of protein synthesis in the sea urchin gonad is discussed.     

INCORPORATION OF AMINO ACIDS INTO SEA URCHIN EGGS STIMULATED INSUFFICIENTLY WITH AN ACTIVATING REAGENT

Investigations were performed on the incorporation of valine-¹⁴C and leucine-³H into sea urchin eggs which were stimulated insufficiently with an activating reagent. Materials used were Pseudocentrotus, Hemicentrotus and Anthocidaris. An insufficient stimulation enhanced the incorporation of amino acids into the eggs, although it provoked no visible cortical changes. The amount of incorporation in this case was 1.6 to 2.7 times as much as that into untreated eggs. In fully activated eggs, the amount of incorporation was more than 4 times that in untreated eggs. The fact that the incorporation of amino acids is increased without accompanying breakdown of the cortical granules indicates that the increase may be linked to an invisible change, probably the fertilizationwave, which is caused by an insufficient stimulation.     

Wound phloem in transition to bundle phloem in primary roots ofPisum sativum L.

Summary 48 hours after interrupting the root stele ofPisum, wound phloem initiated (proximally or distally to the wound) to reconnect the vascular stumps was found to contain some nucleate wound-sieve elements. At the elongating end of an incomplete wound-sieve tube these elements exhibit a sequence of ultrastructural changes as known from protophloem-sieve tubes. Elongation occurs by the addition of newly divided (wound-) sieve-element/companion-cell complexes. In order to dedifferentiate and assume a new specialization formerly quiescent stelar or cortical cells require at least one (mostly more) preliminary division. Companion cells are consequently obligatory sister cells to wound-sieve elements.By reconstruction using serial sections it could be shown that wound-sieve tubes elongate bidirectionally, starting in an early activated procambial cell of the stele. The elongation is directed by the existence of plasmodesmata, preferably when lying in primary pit fields, and by the plane of preceding divisions. Thus, the developing wound-sieve tube can deviate from the damaged bundle and radiate into the cortex as soon as the plane of the preceding divisions is favourable. In the opposite direction, elongating wound-sieve tubes run parallel to pre-existing phloem traces, thus broading their base at the bundle for the deviating part of the wound-sieve tube. Frequently an individual wound-sieve tube is supplemented at the bundle by a further wound-sieve tube which is partly running parallel to it. Both sieve tubes are interlinked with sieve plates by three-poled sieve elements.Ultrastructurally, the developmental changes of nucleate wound-sieve elements follow the known pattern. In spite of its contrasting origin and odd shape a mature wound-sieve element eventually has the same contents as regular sieve elements: sieve-element plastids, mitochondria, stacked ER and small amounts of P-protein within an electronlucent cytoplasm.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.