scholarly journals Quantal tektin synthesis and ciliary length in sea-urchin embryos

1989 ◽  
Vol 92 (3) ◽  
pp. 403-413 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Sea Urchin ◽  
De Novo ◽  
Specific Activity ◽  
High Specific Activity ◽  
Strongylocentrotus Droebachiensis ◽  
De Novo Synthesis ◽  
Structural Element ◽  
Large Pool ◽  
And Control ◽  
Structurally Stable

Previous work using pulse-chase labelling of embryos from the sea-urchin Strongylocentrotus droebachiensis during ciliogenesis, regeneration or steady-state maintenance and elongation showed that a ciliary outer doublet microtubule-associated protein, originally termed component-20, was synthesized in a fixed or quantal amount. This suggested that the limited synthesis of component-20 might limit ciliary length, since the embryo has a large pool of most other ciliary components. Labelling experiments with S. purpuratus embryos now confirm quantal synthesis of component-20, while antibodies to S. purpuratus sperm flagellar tektins identify component-20 as the ciliary equivalent of the flagellar 55 × 10(3) Mr tektin, tektin A. Sequential pulse-chase labelling at various times prior to isolation of cilia proves that the high specific activity of this protein truly reflects de novo synthesis of a structurally stable protein and not rapid protein turnover. Embryos may be animalized by growth in the presence of zinc ions, resulting in cilia averaging nearly twice the normal 20 microns length. When these embryos are pulse-chase labelled during ciliary growth and elongation, labelling of tektin A is proportional to the greater ciliary length, as is the pool of labelled but unincorporated tektins and other minor proteins. Deciliated animalized and control embryos, pulse-chase labelled during their identical phases of ciliary regeneration, incorporate labelled tektin A to the same extent and have similar pools of unincorporated proteins. The correlation of enhanced tektin A synthesis with increased ciliary length and the coincidence of tektin A synthesis with ciliary elongation are observations consistent with the hypothesis that tektin A is a ciliary length-limiting structural element.

Rates of de novo Synthesis of Malate Synthase and Albumins during the Very Early Phase of Germination

1979 ◽  
Vol 34 (12) ◽  
pp. 1237-1242 ◽  
Author(s):  
Wolfram Köller ◽  
Helmut Kindl
Keyword(s):  
Early Phase ◽  
De Novo ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Malate Synthase ◽  
Polyacrylamide Gel ◽  
De Novo Synthesis ◽  
Albumin Fraction ◽  
Average Value ◽  
Large Pool

Abstract Malate synthase is synthesized de novo in the very early phase of germination. Its molecular and immunological properties do not differ from those of malate synthase from fully developed cotyledons. Radioactive leucine was administered to dry seeds of cucumber, and its incorporation into proteins of cotyledons was examined after 2 days of germination. The specific radioactivity of malate synthase, purified by immunoprecipitation and electrophoresis on polyacrylamide gel, was only 1/20 the average value of the total albumin fraction. The minimal incorporation documented by the comparatively low specific activity of isolated malate synthase is discussed in relation to the large pool of malate synthase already present in dry seeds.

scholarly journals Sphingolipid Profiling Reveals Different Extent of Ceramide Accumulation in Bovine Retroperitoneal and Subcutaneous Adipose Tissues

Metabolites ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 473
Author(s):  
Yue Hei Leung ◽  
Sonja Christiane Bäßler ◽  
Christian Koch ◽  
Theresa Scheu ◽  
Ulrich Meyer ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
De Novo ◽  
Specific Activity ◽  
Multiple Reaction Monitoring ◽  
De Novo Synthesis ◽  
Bioactive Lipids ◽  
Tissue Specific ◽  
Sphingosine 1 Phosphate ◽  
Ceramide Accumulation ◽  
Subcutaneous Adipose

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.

scholarly journals Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

1979 ◽  
Vol 82 (3) ◽  
pp. 742-754 ◽  
Author(s):  
C A Gabel ◽  
E M Eddy ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Specific Activity ◽  
High Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Regional Differentiation ◽  
Specific Cell ◽  
Sperm Surface ◽  
Erythrocyte Surface ◽  
Triton X

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

De novo Synthesis of Transfer and 55 RNA1f in Cleaving Sea Urchin Embryos

1972 ◽  
Vol 239 (89) ◽  
pp. 51-53 ◽  
Author(s):  
ANNE F. O'MELIA ◽  
CLAUDE A. VILLEE
Keyword(s):  
Sea Urchin ◽  
De Novo ◽  
De Novo Synthesis ◽  
Sea Urchin Embryos

Enhanced RNA and protein synthesis in adipose tissue of rats adapted to periodic hyperphagia

1968 ◽  
Vol 46 (6) ◽  
pp. 903-906 ◽  
Author(s):  
L. Kazdová ◽  
T. Braun ◽  
P. Fábry ◽  
R. Poledne
Keyword(s):  
Protein Synthesis ◽  
Adipose Tissue ◽  
Rna Synthesis ◽  
De Novo ◽  
Specific Activity ◽  
De Novo Synthesis ◽  
Experimental Conditions ◽  
Rna And Protein Synthesis ◽  
Meal Feeding ◽  
The Difference

RNA synthesis measured by the incorporation of orotic acid-6-14C into RNA was investigated in isolated adipose tissue of control rats and of rats adapted to periodic hyperphagia, evoked by meal-feeding (a single 2-h meal per day). Both groups were fasted for 22 h and subsequently fed a measured test meal for another 2 h. It was revealed that 2 and 4 h after feeding there was no significant change in comparison with values during fasting, whereas in tissue of meal-fed rats the specific activity of RNA gradually increased by 22% and 41% respectively. The difference between controls and meal-fed rats was even much more marked if the specific activity of RNA in fat cells, isolated after incubation of the tissue, was measured. A significantly greater response of meal-fed rats was found when protein synthesis and lipogenesis in adipose tissue were assessed under the same experimental conditions. The possibility is discussed that the enhanced RNA and protein synthesis in adipose tissue of meal-fed rats is associated with de novo synthesis of enzymes involved in adaptive hyperlipogenesis.

scholarly journals Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

1997 ◽  
Vol 8 (11) ◽  
pp. 2187-2198 ◽  
Author(s):  
Raymond E. Stephens
Keyword(s):  
Steady State ◽  
Sea Urchin ◽  
Molecular Chaperone ◽  
De Novo ◽  
Selective Inhibition ◽  
Strongylocentrotus Droebachiensis ◽  
Protein Labeling ◽  
Architectural Proteins ◽  
Ciliary Protein ◽  
Chaperone Hsp70

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [3H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.

scholarly journals DRES-09. REGULATORY EFFECTS OF THE CILIARY GTPASE ARL13B ON PURINE METABOLISM IN GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi73-vi73
Author(s):  
Miranda Saathoff ◽  
Jack Shireman ◽  
Eunus Ali ◽  
Cheol Park ◽  
Issam Ben-Sahra ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Specific Activity ◽  
Purine Metabolism ◽  
P Value ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Double Strand Breaks ◽  
Purine Nucleotides ◽  
Regulatory Effects

Abstract Glioblastoma (GBM) is the most common form of adult primary brain cancer. Despite an aggressive treatment regimen – surgical resection, irradiation, and temozolomide (TMZ) chemotherapy – patients’ prognosis is still grim. TMZ acts by methylating purines, specifically at the O6 and N7 positions of guanine, to induce cytotoxic DNA double-strand breaks. We thus wanted to explore how purine metabolism may contribute to TMZ-resistance. In mammalian cells, purine nucleotides can be recycled by the salvage pathway or generated via de novo synthesis. The salvage pathway is energetically inexpensive relative to de novo thus, highly proliferative GBM cells preferentially utilize the salvage pathway. We have shown that salvage synthesis is reduced in response to TMZ (p-value=0.0021), hinting that the cells may utilize de novo to evade therapy induced alkylation of purines. Using immunoprecipitation-mass spectroscopy analysis, we found a novel interaction between the ciliary GTPase ARL13B and IMPDH2, the rate-limiting enzyme in de novo synthesis. We have shown that this interaction, occurring at the C-terminal domain of ARL13B, plays a significant role in the regulation of purine biosynthesis as abolishing it through ARL13B knockdown reduced flux through de novo (p-value< 0.0001) synthesis as measured by the specific activity of IMPDH2. Further, the lentiviral-mediated rescue of ARL13B brings IMPDH2 activity back to basal levels (p< 0.0001). Given its canonical function as a GTPase, we hypothesize that ARL13B acts as a novel regulator of de novo synthesis by sequestering GDP, allowing IMPDH2 to sense and respond to the cytosolic levels of guanine nucleotides. Without ARL13B the de novo pathway is halted, forcing the cells to rely on salvage to replenish nucleotide pools. Reliance on this pathway in the presence of TMZ causes cells to incorporate damaged nucleotides as a result of the drug’s alkylating action leading to the increased therapeutic efficacy of TMZ.

Structural and Metabolic Interrelationships Among Glycerophosphatides of Rat Liver In Vivo

1971 ◽  
Vol 49 (12) ◽  
pp. 1347-1356 ◽  
Author(s):  
B. J. Holub ◽  
A. Kuksis
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Specific Activity ◽  
Acyl Transfer ◽  
De Novo Synthesis ◽  
Individual Molecular Species ◽  
Relative Specific Activity ◽  
Proportional Contribution ◽  
Specific Activities

The specific activities of individual molecular species of rat liver diacylglycerylphosphorocholine (PC), diacylglycerylphosphoroethanolamine (PE), and diacylglycerophosphorylinositol (MPI) were determined and compared following intravenous injection of glycerol-14C. PC, PE, and MPI contained 41, 51, and 83%, respectively, tetraenoic species, and 40,17, and 9% combined mono-, di-, and trienoic species. The rest of the phosphatide mass of PC, PE, and MPI was contributed by 18, 32, and 8% penta- and hexaenoic species, respectively. The proportions of chemical classes of the glycerophosphatides differed by 1.1- to 18-fold while the fatty acid associations within the unsaturation classes common to these phosphatides varied 2.2- to 17-fold. After 5 min exposure to radioactive glycerol, the mono-, di-, and trienoic species of the PC, PE, and MPI possessed 13–18, 15–50, and 6–42 times, respectively, the specific activity of the tetraenes of the corresponding phosphatide classes. While the pentaenoic and hexaenoic species of PC and MPI had specific activities three to five times those of the respective tetraenes, the higher polyenes of PE were considerably more radioactive and approached the specific activity of the dienoic species of this phosphatide. With progressing time up to 60 min, the tetraenoic species of PC, PE, and MPI showed increases in relative specific activity of 50, 64, and 109%, respectively, in the three phosphatides. These results are consistent with an effective de novo synthesis of the oligoenoic species and a transacylation of the tetraenoic species of all liver glycerophosphatides tested. The proportional contribution of de novo synthesis in comparison to acyl transfer is apparently greater to the formation of PC and PE than to that of MPI.

The occurrence of a soluble glucose 6-phosphatase in cotyledon tissue of Phaseolus vulgaris during germination

1969 ◽  
Vol 47 (7) ◽  
pp. 685-689 ◽  
Author(s):  
J. E. Thompson
Keyword(s):  
Endoplasmic Reticulum ◽  
Phaseolus Vulgaris ◽  
De Novo ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
De Novo Synthesis ◽  
Soluble Enzymes ◽  
Ph Profiles

Cotyledon tissue from seedlings of Phaseolus vulgaris at various stages of germination was homogenized and fractionated under isotonic conditions. Measurement of glucose 6-phosphatase (D-glucose 6-phosphate phosphohydrolase, EC 3.1.3.9) activity in the isolated fractions showed an increase in the level of soluble enzyme with advancing age of the cotyledons, such that by late senescence (10 days of age) approximately 90% of the recovered glucose 6-phosphatase was present in the soluble fraction, and its specific activity was about twofold greater than that of the homogenate. The pH profiles of the bound and soluble enzymes were found to be closely similar. The presence of a soluble glucose 6-phosphatase has been interpreted as indicating either de novo synthesis of the soluble form of the enzyme with advancing senescence, or solubilization of normally bound enzyme which may in turn reflect a partial breakdown in the structure of endoplasmic reticulum in senescent cells.

Induction of the xylitol dehydrogenase of Pullularia pullulans

1988 ◽  
Vol 34 (2) ◽  
pp. 107-111 ◽  
Author(s):  
Juliet K. Sugai ◽  
L. A. Veiga
Keyword(s):  
Enzyme Induction ◽  
De Novo ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Xylitol Dehydrogenase ◽  
Lag Phase ◽  
Differential Rate ◽  
De Novo Synthesis ◽  
Induction Study ◽  
Glycerol Medium

Pullularia pullulans, a yeastlike fungus, metabolizes xylitol via an intracellular xylitol dehydrogenase. Formation of the enzyme was induced by D-xylose and inhibited by cycloheximide, suggesting de novo synthesis. The induction study was carried out with noninduced cells of P. pullulans harvested from a glycerol medium. The cells initiated synthesis of xylitol dehydrogenase without a detectable lag phase when 1.5% xylose was added. The differential rate of xylitol dehydrogenase synthesis was 1.4 units per mg of synthesized protein. Kinetic studies of enzyme induction in a growing culture showed that the highest level of specific activity was reached at 25 to 27 h of growth, with an increase of 200 times over the basal level in the glycerol medium.

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