Thermal compensation in protein and RNA synthesis during the intermolt cycle of the American lobster, Homarus americanus.

1. The in vitro rates of incorporation of precursors into protein and RNA and the concentration of RNA were measured in tissues of intermolt and premolt lobsters acclimated to 5 degrees C and 20 degrees C. Midgut gland, abdominal muscle and gill of intermolt lobsters respond to temperature acclimation by a compensatory translation of the rate-temperature (R-T) curves with respect to the rates of incorporation of 3H-leucine and 3H-uridine into the acid-insoluble fraction. Midgut gland and muscle of premolt animals exhibit either no compensation or inverse compensation; gill tissue exhibits a rotation of the R-T curve. 2. The existence of the complete de novo pathway of pyrimidine biosynthesis is demonstrated in the class Crustacea. NaH14 CO2 is incorporated into orotic acid and orotic-14 C-acid is incorporated into the acid-insoluble fraction. 3. Both the concentration of RNA and the rates of incorporation of precursors of both the salvage and de novo pyrimidine pathways are enhanced in the midgut gland of premolt lobsters, relative to intermolt tissue, under conditions of warm-acclimation.     

Influence of molt cycle and beta-ecdysone on protein synthesis in the chelicerate arthropod, Limulus polyphemus.

1. The effect of molt cycle stage and beta-ecdysone on protein synthesis in the horsehoe crab, Limulus polyphemus, was examined. 2. A pronounced decline in protein specific radioactivity after incubation with 14C-leucine was noted in muscle, midgut gland and operculum from postmolt to intermolt to premolt and in gut and gill tissue from intermolt to premolt. 3. beta-Ecdysone injections produced an early stimulation of protein synthesis in the midgut gland followed by strong inhibition within 48 hr. 4. Results are compared with those obtained in mandibulate arthropods.     

Ca2+-Activated Force Production and Calcium Handling by the Sarcoplasmic Reticulum of Crustacean Muscles During Molt-Induced Atrophy

Growth in crustaceans is an intermittent process centered aroundthe principal event of ecdysis. A major problem facing decapodcrustaceans at the time of ecdysis is the withdrawal of thelarge muscle mass of the chelae through the narrow basi-ischialjoints. To overcome this problem the muscle undergoes an atrophytriggered by the molt, which reduces the muscle mass. Once theanimal is freed from the old exoskeleton, the muscle fibers,must elongate to accommodate the new larger exoskeleton. Despitethis major myofibrillar remodification, the muscles are thoughtto remain functional over the molt cycle. Studies using skinnedmuscle fibers have shown that long-sarcomere fibers maintaintheir function over the molt cycle while the contractile propertiesof the short-sarcomere fibers are modified, as fibers couldnot withstand maximal activation with Ca²⁺ during the premoltstage. In this study the maximum Ca²⁺-activated force productionand the ability of the sarcoplasmic reticulum (SR) to releaseaccumulated Ca²⁺ has been investigated in the two major fibertypes in the claw muscle of Cherax destructor, in the stagesjust prior to ecdysis and during inter molt. In both long- andshort-sarcomere fibers, the amount of Ca²⁺ released by the SRwas not different in premolt and intermolt stages. However,the maximum releasing capacity of the SR was reached in a shortertime during the premolt suggesting that Ca²⁺ is being accumulatedat a faster rate. The force production was greatly reduced andwas graded during the premolt in both fiber types. This modulationof force appears to be the most likely candidate regulatingthe magnitude of the force development in the periods when fibersare undergoing myofibrillar remodification and thus may serveto prevent fiber damage.     

The Foregut of Gecarcinus lateralis as an Organ of Salt and Water Balance

The permeability of the foregut of the land crab, Gecarcinuslateralis, to tritiated water (THO), Na²², and Cl³⁶ was studiedin vitro during the intermolt period and after ecdysis. In crabswith eyestalks, the foregut is permeable to water and ions inthe direction hemolymph-to-lumen and lumen-to-hemolymph, bothduring the intermolt period and after ecdysis. However, theforegut of animals without eyestalks is impermeable after ecdysis. The movement of THO always follows the movement of Na²² acrossthe wall of the foregut, while the movement of Cl³⁶ may or maynot be correlated with the movement of Na²² and THO. Comparisonof the ratio of water to ions in the hemolymph with the ratiocalculated from radioisotope flux indicates that Na⁺ and waterare probably moving isosmotically, although not necessarilyaccompanied by Cl^– When an extract of the thoracic ganglionic mass of G. lateralisis added to the "hemolymph side" of the foregut in vitro, thereis immediately a large increase in permeability to water andsalts. This occurs in the foregut of crabs with eyestalks duringintermolt and also in eyestalkless crabs after ecdysis. Thus, not only is the foregut of Gecarcinus lateralis permeableto water and salts in both directions, but also the extent ofits permeability is under neuroendocrine control. As a consequence,the animal may be able to move water and salts into the foregutor out of it into the hemolymph as needed. This may be an importantadaptation for a terrestrial crab that must conserve water,especially at the critical time of ecdysis.     

Role of eyestalk hormone in the carbohydrate metabolism of a marine penaeid prawn,Parapenaeopsis hardwickii (MIERS) (Crustacea,Decapoda, Penaeidae)

Data furnished here concern with the role of eyestalk hormone in the regulation of carbohydrate metabolism in Parapenaeopsis hardwickii. Bilateral eyestalk ablation has brought about a significant (P < 0.01) fall and rise in the glycogen content in the midgut gland and abdominal muscle respectively. Although eyestalk ablation resulted in a significant (P < 0.01) depletion of fat in midgut gland, n0 significant (P > 0.05) change was observed in the abdominal muscle. Eyestalk extract administration in eyestalk-less prawns has significantly (P < 0.05) restored the glycogen and fat metabolites in the midgut gland. There was an obvious change in the glycogen content of the midgut gland and abdominal muscle of normal prawns when injected with eyestalk extracts from prawns in different molting stages. Eyestalk extract from intermolt prawns caused a significant (P < 0.05) decrease and increase in the glycogen quantity in the midgut gland and abdominal muscle respectively. Eyestalk extract from premolt and postmolt prawns has, although not significantly (P > 0.05), decreased and increased the utilization of glycogen respectively in the midgut gland. The physiological significance of these findings are discussed briefly.Paper forms part IV of the series

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Elongation factor 1Bgamma (eEF1Bgamma) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii

Eukaryotic elongation factor 1Bγ (eEF1Bγ) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bγ has properties which suggest sensory or regulatory activities. We have cloned eEF1Bγ from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bγ and 65% identity to Artemia salina eEF1Bγ. We measured eEF1Bγ expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bγ expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bγ expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bγ was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bγ expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bγ were found after 1 week of acclimation to 4 °C. These results show that eEF1Bγ is regulated at the mRNA level with tissue-specific differences in expression patterns.     

Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast.

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.     

Mobilization of carbohydrates in tissues of female silkworms, Bombyx mori, during metamorphosis

The metabolic activity and mobilization of carbohydrates among tissues of female silkworms were examined during metamorphosis by injecting radioactive ¹⁴C-glucose as a tracer. The isotope injected was incorporated into various tissues with varying degrees and reached a relatively stable state in all tissues tested in about 240 min. The metabolic activities analysed by 4 hr pulse labelling were different for different tissues and ages; in glycogen synthetic activity midgut was highest on the day of the larval-pupal ecdysis, the fat body 2 days later, and ovaries a further 4 days later.When the isotope was injected on the day of larval-pupal ecdysis, it was found predominantly in glycogen first in the midgut, then in the fat body, and finally in the ovaries, proceeding through development. The total radioactivity recovered in the glycogen fractions from these tissues was almost constant throughout development. Ovariectomy caused a rise in synthesis of both glycogen and trehalose in the fat body during the second half of development.From these results it is proposed that the dermand of developing ovaries for carbohydrates exerts a controlling influence over mobilization of glycogen in the fat body.     

Assessment of biomarkers of protein anabolism in skeletal muscle during the life span of the rat: sarcopenia despite elevated protein synthesis

Loss of muscle strength is a principal factor in the development of physical frailty, a condition clinically associated with increased risk of bone fractures, impairments in the activities of daily living, and loss of independence in older humans. A primary determinant in the decline in muscle strength that occurs during aging is a loss of muscle mass, which could occur through a reduction in the rate of protein synthesis, an elevation in protein degradation, or a combination of both. In the present study, rates of protein synthesis and the relative expression and function of various biomarkers involved in the initiation of mRNA translation in skeletal muscle were examined at different times throughout the life span of the rat. It was found that between 1 and 6 mo of age, body weight increased fourfold. However, by 6 mo, gastrocnemius protein synthesis and RNA content per gram of muscle were lower than values observed in 1-mo-old rats. Moreover, the relative expression of two proteins involved in the binding of initiator methionyl-tRNA to the 40S ribosomal subunit, eukaryotic initiation factors (eIF)2 and eIF2B, as well as the 70-kDa ribosomal protein S6 kinase, S6K1, was lower at 6 mo compared with 1 mo of age. Muscle mass, protein synthesis, and the aforementioned biomarkers remained unchanged until approximately 21 mo. Between 21 and 24 mo of age, muscle mass decreased precipitously. Surprisingly, during this period protein synthesis, relative RNA content, eIF2B activity, relative eIF2 expression, and S6K1 phosphorylation all increased. The results are consistent with a model wherein protein synthesis is enhanced during aging in a futile attempt to maintain muscle mass.     

Gene expression patterns, and uptake and fate of fed ABA in white spruce somatic embryo tissues2

Changes in cellular protein accumulation and in in vivo andin vitro protein synthesis, in somatic embryo tissues of whitespruce during a 42 d maturation period were followed by two-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-DSDS-PAGE). These investigations were complemented by an analysisof uptake and fate of fed abscisic acid (ABA) in somatic embryotissues grown on maturation medium. When Stage 1 somatic embryoswere cultured on ABA-containing maturation medium, many changeswere observed in patterns of gene expression and in proteinsynthesis and accumulation which could be associated with embryodevelopment. The polypeptides observed could be categorizedas constitutive, embryo-abundant, embryo maturation-relatedand embryo stage-related, as well as those with non-specificchanges. Accumulation of label from fed ³H-(+)-ABA in embryotissues reached a plateau 3 d after Stage 1 somatic embryoswere placed on maturation medium. ABA taken into tissues wasrapidly metabolized, and 40% of radioactivity in tissues after1 d of culture resulted from ABA metabolites. This value increasedto 90% after 3 weeks culture. Conjugated ABA and oxidized ABA(phaseic acid and dihydrophaseic acid) were major forms of ABAmetabolites in spruce embryo tissues. Using a single 42 d cultureperiod following transfer to medium with ABA, the conditionsthat stimulate the sequence of developmental changes of somaticembryo maturation during the first 21 d do not reoccur duringthe second 21 d. Unless greater synchronization of Stage 1 culturescan be achieved, it is therefore unlikely that yields of maturesomatic embryos will be increased by this method. Key words: Abscisic acid, gene expression, Picea glauca (Moench) Voss, protein synthesis, somatic embryo maturation     

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; )     

L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper (Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70μl/h and reabsorbed across the pharate pupal midgut at about 26μl/h. L-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850nmol about 8h before pupal ecdysis (P-8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P相似文献   

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.