Regulation of Protein Synthesis in Embryos of Ilyanassa obsoleta

Much of the protein synthesis during early development in Ilyanassaresults from the translation of oogenetic mRNA. We show thatmicrotubule proteins are products of this translation, and thattheir synthesis is subject to translation level regulation.We also show that translation level regulation is involved inthe function of the polar lobe by making comparisons of theelectrophoretic patterns of synthesis of ¹⁴C labeled proteinsof normal embryos with the patterns of synthesis of ³H labeledproteins of embryos from which the polar lobes had been removedat the trefoil stage. Controls utilizing biochemical and morphologicalmarkers were performed to assure that normal and delobed embryoswere developing at equivalent rates. The expression of significantdifferences in the patterns of protein synthesis were foundbetween normal and delobed embryos, and these differences werenot dependent upon concomitant RNA synthesis. These differenceswere observable as early as after only 24 hours of development,although organogenesis does not begin until much later in development.Therefore, the observed differences probably reflect determinativeevents. The results support the hypothesis that the developmentaldeterminants of the polar lobe may include specific, preformedmRNA sequences, or specific regulators of translation.     

In vivo Studies on Protein Synthesis in Developing Seeds of Canavalia gladiata D.C.

In successive stages of seed development of Canavalia gladiata,fruits were harvested and time-course changes of seed proteinaccumulation and SDS-gel electrophoretic patterns of the polypeptideswere examined. Albumin content increased gradually after thestage of 1.8 g fresh weight per seed, reached a peak, then decreasedat later stages. SDS-gel electrophoresis showed a clear changein albumin composition between the stages of 2.3 g and 3.6 gfresh weight per seed. Seed globulins were synthesized activelyafter the stage of 1.8 g fresh weight per seed, and the majorpolypeptides of seed globulins, including the main polypeptidesof canavalin, were first detected at this stage by gel electrophoresis.In contrast to the albumins, these polypeptides of globulinscontinued to accumulate until the maturation was completed.In vivo protein synthesis was also analyzed by SDS-gel electrophoresisand fluorography, after [³⁵S]methionine had been fed to cotyledonsections of maturing seeds at the stages of 3.4 g and 5.0 gfresh weight per seed. The results indicated that the synthesisof canavalin starts earlier than that of concanavalin A andalso ends earlier during seed development. (Received December 10, 1985; Accepted June 2, 1986)     

Changes in mimosa epicotyl soluble nucleic acid patterns: Comparisons between changes related to growth rate and changes related to degree of maturation

Soluble nucleic acids from Albizzia julibrissin epicotyl tissueswere extracted and fractionated at different times during lategermination. Seedlings were grown on two different growth regimes,one with a relatively constant growth rate and an increasingpercentage of mature tissues, and a second with an increasinggrowth rate but relatively constant percentage of mature tissues.Consistent changes were observed in distribution of specificsoluble nucleic acid fractions with growth rate and stage ofdevelopment. Stability of terminal groups of soluble nucleicacid also were related to growth and developmental patterns.Finally, degree of amino acid acceptance by total tRNA decreasedwith stage of development, but did not relate to growth ratesor percentage of mature tissues. ¹Contribution from the Missouri Agricultural Experiment Station.Journal Series No. 5586. (Received February 3, 1969; )     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Adenosine 5'-Phosphosulf ate Sulfotransferase from Euglena: Enzyme-Bound Intermediates

Adenosine 5'-phosphosulfate sulfotransferase (APSST) purifiedfrom Euglena gracilis Klebs var. bacillaris mutant W₁₀BSmL byammonium sulfate precipitation, Sephadex G-100 gel filtration,reactive blue agarose, reactive dye agarose and DEAE-cellulosecan be labeled by incubation with AP³⁵S and separated from smallradioactive compounds on Sephadex G-50. Most of the label isnot exchangeable with nonradioactive APS and therefore is notassociated with bound substrate. On non-inactivating SDS-PAGE,a radioactive band at the position of native APSST tetramershows APSST activity (measured as acid-volatile radioactivity).Labeled protein hydrolyzed with Pronase yields radioactive S-sulfocysteine,indicating that at least one cysteine residue of APSST acceptsa sulfo group from APS to form E-S-SO₃^–. A labeled lowmolecular weight compound can be separated from the proteinby paper electrophoresis or by treatment with acidic proteindenaturing reagents such as trifluoroacetic acid (TFA) or trichloroaceticacid (TCA). This labeled compound (perhaps the sulfo-carrier)behaves as a strong acid on paper electrophoresis and is stabilizedby iodoacetamide or acidic conditions but degrades to thiosulfate,sulfate and other compounds as the pH is raised. The radioactivityin APSST is exchangeable with sulfite or thiosulfate. AMP inhibitsAPSST in the formation of acid-volatile radioactivity by competingwith APS, but APA inhibits APSST activity uncompetitively. AK_m of 0.1 µM for APS and K_i of 0.1 mM for AMP and 0.6mM for APA are obtained when a saturating amount of dithiothreitol(DTT) is used as the thiol. A mechanism is proposed for theinitial reaction(s) catalyzed by APSST. ¹Present address: Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, U.S.A.     

The Effect of Growth in D2O on the Metabolism of Winter Rye

The metabolism of winter rye seedlings (Secale cereale, L. ev.Winter) cultured in 99.8 per cent D₂O was investigated. Comparedwith water-grown seedlings, the protein content was much lowerin the D₂O-cultured seedlings and the pattern of incorporationof [³H]leucine and [³H]phenylalanine into protein was substantiallydifferent. Seedlings cultured in D₂O incorporated [³H]thymidineinto DNA, but did not take up [³H]uridine. The results suggestthat some of the toxic effects of D₂O culture on higher plantscan be attributed to a partial block of protein synthesis.     

Studies on nucleic acids in plastids of the pigment mutant C-2A{acute} of Scenedesmus obliquus

Pigment mutant C-2A{acute} of Scenedesmus obliquus whose chlorophyllformation and chloroplast development are light dependent, wasstudied for the nucleic acid content of its plastids. The ribosomalRNA of plastids of the achlorophyllous or greened mutant C-2A{acute},did not show any difference from that of the wild type. Incorporationof [5-³H] uridine into mutant cells was partially inhibitedby rifampicin, indicating this part as being plastidial incorporation.Since there were no significant differences in the ribosomalRNA of plastids between the mutant and the wild type of Scenedesmus,the ribosomal system in the plastids of mutant C-2A' seemednot to be affected by the mutation. C_sCl gradient patterns ofScenedesmus mutant and wild-type DNA were almost identical withthose of Chlorella DNA. A peak at a buoyant density of 1.69g/cm³, the same as that of Chlorella chloroplast DNA, couldbe identified in Scenedesmus also as plastid DNA because itdisappeared after prolonged treatment with myxin and hybridizedwith rifampicin-sensitive pulse-labelled RNA. This peak waspresent to nearly the same degree in the mutant and the wildtype, indicating that a larger deficit of plastid DNA did notoccur in the mutant. Whether or not the mutation might be localizedin the plastid genome is discussed. (Received March 19, 1976; )     

Amylolytic Activity and Carbohydrate Levels During the Stamen Ontogeny of a Male Fertile, and a 'Gibberellin-Sensitive' Male Sterile Mutant of Tomato (Lycopersicon esculentum)

The activity of amylases, and the level of starch and solublesugar content were analysed in the normal (+/+), a ‘gibberellin-sensitive’male sterile stamenless-2 (sl-2/sl-2) mutant, and GA³-revertedsl-2/sl-2 mutant stamens of tomato (Lycopersicon esculentumMill.), at various stages of development. In the mutant stamens,amylolytic activity did not differ from that of the normal untilthe tetrad stage, but thereafter it was significantly lowerthan that of normal stamens. The starch content also did notdiffer in the two lines at early stages of development. However,at later stages it decreased in normal stamens, whereas it remainedunchanged in the mutant. The soluble sugar content graduallyincreased during the development of normal stamens. But in mutantstamens, it remained the same throughout development and wassignificantly lower than the normal. In GA₃-reverted mutantstamens, the amylolytic activity and the level of starch andsoluble sugars were comparable to normal stamens. It is proposedthat the sl-2/sl-2 mutation, through its effects on endogenousgibberellins, affects the activity of amylases which, in turn,result in lower sugar levels leading ultimately to abnormalpollen development. Key words: Amylases, male-sterility, tomato     

MORPHOGENESIS IN SPIEODELA OLIGORRHIZA: EFFECTS OF PYRIMIDINE BASE ANALOGUES ON INITIATION, ELONGATION AND DIFFERENTIATION OF FRONDS

The effect of four pyrimidine base analogues and their antidoteson S. oligorrhiza was studied. FUdR stopped cell division at concentrations of 4 10^–7M and higher. This effect could be nullified by the additionof 4 10^–6 M thymidine. Neither uridine nor uracil hadan antidotal effect on FUdR. FU (8 10^–6 M or higher concentration) affected celldivision, frond elongation and differentiation, and could notbe counteracted by either thymidine or uracil. TU (8 10^–4 M) rather specifically inhibited differentiationof frond tissues, while not preventing cell division or theinitiation of new generations. Uracil and uridine at about equimolarconcentrations completely counteracted the TU effect. AzU (10^–3 M) suppressed cell division, frond elongationand frond differentiation. When thymidine (10^–3 M) wasadded simultaneously with AzU only cell elongation and differentiationof fronds were inhibited, but cell division proceeded. 10^–3M uracil (but not uridine) counteracted all effects of AzU. ¹ Based on a portion of the senior author's Ph.D. Thesis.     

Variations in the Chilling Sensitivity of Suspension-Cultured Cells of Mung Bean (Vigna radiata [L.] Wilczek) during the Growth Cycle

Variations in the chilling sensitivity of mung bean (Vigna radiata[L.] Wilczek) cells in suspension culture were studied withreference to the growth cycle. At the initial stages after subculture,cells were relatively insensitive to chilling, but chillingsensitivity increased abruptly thereafter and reached a maximumat the early stage of exponential growth, namely 6 days aftersubculture. Upon further culturing, cells became chilling-tolerant,becoming most tolerant at the late stage of exponential growth,namely 14–15 days after subculture. The results of thereciprocal exchange of conditioned culture medium between thecells at the early and the late exponential phases of growthprior to cold incubation revealed that the change in sensitivitywas due primarily to changes in physiological features of cellsand not to changes in the chemical composition of the culturemedium. In support of this possibility, we observed that thecold sensitivity of the tonoplast H⁺-ATPase in vivo changedmarkedly as a function of growth cycle: it was very sensitiveat the early stage of exponential growth and less sensitiveat the late stage. ¹Contribution no. 3668 from The Institute of Low TemperatureScience, Hokkaido University.     

Mitochondrial alterations in human gastric carcinoma cell line

We compared mitochondrial function, morphology, and proteome in the rat normal gastric cell line RGM-1 and the human gastric cancer cell line AGS. Total numbers and cross-sectional sizes of mitochondria were smaller in AGS cells. Mitochondria in AGS cells were deformed and consumed less oxygen. Confocal microscopy indicated that the mitochondrial inner membrane potential was hyperpolarized and the mitochondrial Ca²⁺ concentration was elevated in AGS cells. Interestingly, two-dimensional electrophoresis proteomics on the mitochondria-enriched fraction revealed high expression of four mitochondrial proteins in AGS cells: ubiquinol-cytochrome c reductase, mitochondrial short-chain enoyl-coenzyme A hydratase-1, heat shock protein 60, and mitochondria elongation factor Tu. The results provide clues as to the mechanism of the mitochondrial changes in cancer at the protein level and may serve as potential cancer biomarkers in mitochondria. two-dimensional gel electrophoresis proteomics; biomarker; cancer     

The Development of Fast-Start Performance in Fishes: Escape Kinematics of the Chinook Salmon (Oncorhynchus tshawytscha)

C-starts are high acceleration swimming movements critical forpredator avoidance by fishes. Since larval fishes are particularlyvulnerable to predation, C-start behavior is likely to be especiallyimportant during early life history stages. This paper examinesthe developmental changes in C-start performance with kinematicdata on immature chinook salmon (Oncorhynchus tshawytscha) (eleuthroembryostage, sensu Balon, 1975). The scaling of C-start kinematicsof immature fishes differs from that of adults. Adult C-startdurations increase with increasing body length while C-startdurations of immature fishes decrease (e.g., adult stage 1 duration[sec] = 0.0019.length [L] [cm] $ 0.026 [R² = 0.77] [Webb, 1978];eleuthroembryos stage 1 duration [sec] = –0.026L [cm]$ 0.100 [R² = 0.81]). Distance traveled during stage 2 alsodiffers between adult and immature fishes. Adult distance traveledscales directly with length (distance [cm] = 0.38L^1.01 [cm],R² = 0.96 [Webb, 1978]) while chinook eleuthroembryo distancetraveled is positively allometric with length (distance [cm]=0.37L¹³¹ [cm], R² = 0.83). There are similarities in the developmentof C-starts and burst swimming. For example, mean velocity scalessimilarly between the two locomotor modes (For burst swimming:U_mean [cm/sec] = 8.1 ± 1.1L [cm] $ 4.89 [R² = 0.86] [Webband Corolla, 1981]. For C-start stage 2: U_mean [cm/sec] = 10.96L[cm] - 14.09 [R² = 0.70]). This study demonstrates that C-startescape performance improves during early post-hatching development.Comparisons of immature chinook salmon fast-starts with dataon larval burst swimming and on adult C-starts suggest thatchanges specific to developing fish affect the scaling of kinematicparameters.     

Heat-Induced Changes in Thermosensitivity and Gene Expression during Development

The thermosensitivity of developing embryos of the fresh water snail Lymnaea stagnalis was investigated from the 4-cell stage to the 3-day-old trochophore larva by means of survival curves for 43.6°C. Cleavage stage embryos were extremely thermoresistant as compared with older stages, and thermosensitivity increases during the development.
Pretreatment with a mild heat exposure (10 min at 39°C) did not induce thermotolerance at the 4-cell stage, but it did so in the early gastrula and trochophora. Development of thermotolerance in 1-, 2-, and 3-day-old stages showed an identical kinetic pattern.
After incubation in ³⁵S-methionine one-dimensional gel electrophoresis was carried out with or without preheating. At the 4-cell stage no enhanced synthesis of heat shock proteins was induced by exposure to heat. At stages of 1 day and older heat induced the enhanced synthesis of the heat shock proteins with apparent molecular weights of 38, 65 and 70 kilodaltons. The synthesis of heat shock protein 70 changes during the early development of Lymnaea both in its constitutive level and in its ability to be enhanced by heat treatment.     

Sectorial Patterns in Leaves on Fruit Tree Shoots Produced by Radioactive Assimilates and Solutions

In vigorously growing shoots of apple and plum ¹⁴C-assimilateswere translocated from a ‘fed’ leaf to particularsectors of other leaves in a distribution pattern associatedwith the phyllotaxis; the same sectorial and distribution patternswere produced by ³²P phosphate solution taken into the shootthrough a cut petiole. The frequency with which a given sectorialpattern occurred at a particular position on the phyllotacticspiral was ascertained. Such patterns were not observed abovethe third rolled leaf in the apple shoot apex. Killing the phloem in the petiole prevented egress of labelledassimilate but not of ³²P solution. Barkringing above the sourceleaf reduced, but did not completely prevent, assimilate movementup the stem, suggesting some translocation in the xylem. Distribution of label from ⁴⁵CaCl₂, ⁸⁶RbCl and [³H]asparagine,incorporated through cut petioles, did not follow the same patternas label from ³²P solutions. Malus pumila Mill., apple, Prunus domestica L., Prunus insititia. L., leaf plum, patterns, transport of radioisotopes, vascular phyllotaxis     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: I. EVIDENCE OF SYNTHESIS IN DIFFERENT PROTEINS AND CELLULAR COMPONENTS

¹⁴C-proline was supplied to aerated potato disks, in which celldivision was occurring, and also to rapidly growing potato carrotexplants. It was absorbed and incorporated into all the subcellularprotein fractions examined, including the electrophoreticallydistinguishable fractions of the soluble protein of the potatodisks and explants. The ¹⁴C-proline was partially convertedto ¹⁴C- hydroxyproline in all the protein fractions, exceptfor one of the soluble protein fractions of potato explantsand the soluble proteins of one set of potato disks. Most ofthe ¹⁴C-proline and ¹⁴C-hydroxyproline contained in the tissuewas found in the soluble protein and also in the cellular fragmentsobtained by centrifugation at 500 g. The relative importanceof the soluble protein in the incorporation of ¹⁴C-proline andits conversion to ¹⁴C-hydroxyproline was greatest over a shortperiod of a few hours of contact with the ¹⁴C-proline supplied.Over a longer period (70 hours) the cellular fragments (500g) had become the most important and contained over 40 per cent.of the total ¹⁴C, and more than 60 per cent. of the ¹⁴C-hydroxyproline,in the protein of the tissues. In the soluble fraction of potatoexplants, seven protein bands were distinguishable on electrophoresis.A different but characteristic value of the ratio ¹⁴C-hydroxyprolineto ¹⁴C-proline was associated with each protein band, exceptfor the one region where ¹⁴C-hydroxyproline did not occur. Thebasic proteins (i.e. those moving towards the cathode) werethe most active in the incorporation of ¹⁴C-proline and itsconversion to ¹⁴C-hydroxyproline. The rather general distributionof the ¹⁴C-hydroxyproline is noted and the possible siginificanceof the basic proteins and the proteins associated with the cellularfragments (500 g) is considered in relation to the growth, celldivision, and cell wall formations which occurs in the rapidlygrowing tissue cultures.     

Protein Synthesis in the Desiccation Tolerant Angiosperm Xerophyta villosa during Dehydration

Leaves of the resurrection plant Xerophyta villosa appear tobecome desiccation tolerant while they dry on the intact plant.After a small decline during moderate water stress, the polyribosomecontent of attached leaves appears to rise at 50% relative watercontent (RWC) to almost double the content in controls, beforeit finally declines to zero at 20% RWC. Partition of leaf solubleprotein by polyacrylamide gel electrophoresis indicates an increasein protein with low mobility in dehydrated leaves. Studies onthe incorporation of ¹⁴C-valine and ³H-valine, and of proteinsdissociated into their component polypeptddes suggest that proteinsynthesis during dehydration is at least partly responsiblefor the changes in soluble protein.     

A Protein from Immature Raspberry Fruits which Inhibits Endopolygalacturonases from Botrytis cinerea and other Micro-organisms

A polygalacturonase-inhibiting protein (PGIP) was purified fromimmature raspberry fruits using ion exchange chromatography.The protein was composed of a single polypeptide chain withM_r of 38·5 kDa and a pI residing above pH 10. Kineticstudies suggested that the inhibition was of a non-competitivenature. The PGIP inhibited two endopolygalacturonases (endo-PG)purified from Botrytis cinerea and an endo-PG produced by Aspergillusniger to varying degrees but did not inhibit two exo-PGs purifiedfrom B. cinerea, bacterial endopectate lyases and bacterialendo-PGs. The concentration of PGIP at various stages of flowerand fruit development was determined. The inhibitor was notdetected in the flower, but reached a maximum of 69 units g^–1in the immature green fruit decreasing to 9 units g^–1as fruits matured. The N-terminal amino-acid sequence was determined. Key words: Polygalacturonase-inhibiting protein, Rubus idaeus, red raspberry, Botrytis cinerea, pectinases     

造血器官机能障碍后家蚕血淋巴中蛋白质成分的变化

为了研究家蚕Bombyx mori造血器官机能障碍后其血淋巴中蛋白质成分的变化,利用重离子射线局部照射家蚕幼虫的造血器官,检测了照射后家蚕血淋巴中的蛋白质成分及注射大肠杆菌后在体内诱导出现的应急蛋白量的变化。结果表明,照射蚕血淋巴中的蛋白质含量与对照蚕之间没有明显的差异。但在成分分析时发现,5龄起蚕血淋巴中70 kD附近的3条蛋白质谱带比对照蚕的浓度要高,随着个体的发育两者的浓度都上升;5龄后期则相反,对照蚕的浓度比照射蚕高;脂肪体中贮藏蛋白质的含量具有相似的变化趋势。用家蚕贮藏蛋白质SP-1及SP-2的抗血清进行免疫印迹反应的结果显示：70 kD附近的3条蛋白质谱带的最上面的一条为贮藏蛋白质SP-1,下面的二条为贮藏蛋白质SP-2;同时照射蚕血淋巴中分子量约为24 kD的蛋白质成分也发生变化,5龄前期的浓度比对照蚕低,5龄第3天几乎检测不到;全体照射与造血器官局部照射蚕之间的结果相似。照射蚕注射大肠杆菌后在体内诱导出现的应急蛋白量明显比对照蚕要少。由此认为家蚕幼虫造血器官与血淋巴中的蛋白质成分有关,造血器官的机能障碍、血球的数量减少可影响脂肪体中蛋白质的合成,从而使存在于血淋巴中的蛋白质成分发生变化。