scholarly journals Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga

1985 ◽  
Vol 231 (3) ◽  
pp. 609-615 ◽  
Author(s):  
D Raederstorff ◽  
M Rohmer
Keyword(s):  
Culture Medium ◽  
De Novo ◽  
Sterol Composition ◽  
Sterol Biosynthesis ◽  
Acanthamoeba Polyphaga ◽  
Biochemical Processes ◽  
Unicellular Algae ◽  
Unknown Structure ◽  
Soil Amoeba

The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells.

scholarly journals Membrane Sterol Composition in Arabidopsis thaliana Affects Root Elongation via Auxin Biosynthesis

2021 ◽  
Vol 22 (1) ◽  
pp. 437
Author(s):  
Meng Wang ◽  
Panpan Li ◽  
Yao Ma ◽  
Xiang Nie ◽  
Markus Grebe ◽  
...  
Keyword(s):  
Root Elongation ◽  
Auxin Transport ◽  
Sterol Composition ◽  
Polar Auxin Transport ◽  
Sterol Biosynthesis ◽  
Auxin Biosynthesis ◽  
Root Tip ◽  
Auxin Signaling ◽  
Auxin Response ◽  
Short Root

Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (β-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.

Characterization of protein production by caprine placental membranes: identification and immunolocalization of retinol-binding protein

1995 ◽  
Vol 146 (3) ◽  
pp. 527-534 ◽  
Author(s):  
K H Liu ◽  
J C Huang ◽  
J D Godkin
Keyword(s):  
Protein Production ◽  
Culture Medium ◽  
De Novo ◽  
Binding Protein ◽  
Yolk Sac ◽  
Retinol Binding Protein ◽  
Minimum Essential Medium ◽  
Placental Membranes ◽  
Extraembryonic Membranes

Abstract Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5·3–6·3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin. Journal of Endocrinology (1995) 146, 527–534

scholarly journals Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 301-306
Author(s):  
M Ogura ◽  
N Tanabe ◽  
J Nishioka ◽  
K Suzuki ◽  
H Saito
Keyword(s):  
Cell Line ◽  
Plasma Protein ◽  
Culture Medium ◽  
De Novo ◽  
Specific Activity ◽  
Calf Serum ◽  
Protein S ◽  
Immunoblot Analysis ◽  
Functional Assay ◽  
Functional Protein

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

scholarly journals Computational design and experimental verification of a symmetric protein homodimer

2015 ◽  
Vol 112 (34) ◽  
pp. 10714-10719 ◽  
Author(s):  
Yun Mou ◽  
Po-Ssu Huang ◽  
Fang-Ciao Hsu ◽  
Shing-Jong Huang ◽  
Stephen L. Mayo
Keyword(s):  
Protein Interactions ◽  
De Novo ◽  
Computational Design ◽  
Atomic Level ◽  
Protein Interfaces ◽  
Unknown Structure ◽  
Α Helix ◽  
Distinct Features ◽  
Novel Protein

Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein–protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by β-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix–mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability.

Exogenous dTMP utilization by a novel tup mutant of Saccharomyces cerevisiae

1982 ◽  
Vol 152 (1) ◽  
pp. 111-119
Author(s):  
L F Bisson ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Culture Medium ◽  
De Novo ◽  
The Other ◽  
Normal Cells ◽  
Pyrimidine Bases

The rate and extent of entry of dTMP were measured in strains of Saccharomyces cerevisiae carrying two new tup mutations (tup5 and tup7) and most of the other tup mutations which have been reported previously by others. The tup7 mutation allowed dramatically greater accumulation of dTMP than any of the other mutations tested. Specific labeling of DNA by [CH3-3H]dTMP, fate of the dTMP pool inside of the cells, and degradation of the dTMP in the culture medium were investigated in strains carrying the tup7 mutation. The extracellular dTMP was not appreciably degraded, and that accumulated intracellularly was readily phosphorylated to dTDP and dTTP. Under optimum labeling conditions, 60 to 80% of the total thymidylate residues in newly synthesized DNA were derived from the exogenously provided dTMP, even in the absence of a block in de novo dTMP biosynthesis. An apparent Km for entry of 2 mM dTMP was found. The tup7 mutation increased permeability to dTMP (and some other 5'-mononucleotides), but did not affect uptake of nucleosides and purine and pyrimidine bases. Uptake of dTMP could be almost completely inhibited by moderate concentrations of Pi. These findings and other observations suggest that entry of dTMP in strains carrying the tup7 mutation is mediated by a permease whose function in normal cells is the transport of Pi.

scholarly journals Sterol biosynthesis in the echinoderm Asterias rubens

1975 ◽  
Vol 146 (1) ◽  
pp. 25-33 ◽  
Author(s):  
A G Smith ◽  
L J Goad
Keyword(s):  
Body Wall ◽  
De Novo ◽  
Mevalonic Acid ◽  
The Body ◽  
Sterol Biosynthesis ◽  
Asterias Rubens ◽  
Nonsaponifiable Lipid ◽  
Major Sterol ◽  
Dietary Origin ◽  
Isolated Tissues

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4α-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol and 4α-methyl-5α-cholest-7-en-3β-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5α-cholest-7-en-3β-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9β,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5α-cholest-7-en-3β-ol.

scholarly journals Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines

1988 ◽  
Vol 250 (2) ◽  
pp. 349-353 ◽  
Author(s):  
E Malvoisin ◽  
F Wild ◽  
G Zwingelstein
Keyword(s):  
Cell Lines ◽  
Cell Types ◽  
Sterol Composition ◽  
Promyelocytic Leukaemia ◽  
Control Analysis ◽  
Leukaemia Cell ◽  
Biochemical Processes ◽  
Variant Cell ◽  
Line Blast ◽  
Resistant Cells

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.

Kinetics of a rapid, luminol dependent chemiluminescence signal induced in HL-60 cells by amphotericin B and other stimulants

1991 ◽  
Vol 69 (9) ◽  
pp. 618-623 ◽  
Author(s):  
D. A. O'Keefe ◽  
D. R. James ◽  
W. R. Ware ◽  
N. O. Petersen
Keyword(s):  
Tissue Culture ◽  
Superoxide Dismutase ◽  
Amphotericin B ◽  
Culture Medium ◽  
Polyene Antibiotic ◽  
Biochemical Processes ◽  
Rate Determining Step ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Peak Value

Addition of the polyene antibiotic amphotericin B or tissue culture medium to nondifferentiated HL-60 cells in the presence of luminol induces a chemiluminescence signal that reaches a peak value within a few seconds and decays exponentially in less than a minute. The kinetics of the signal and its modulation by superoxide dismutase, catalase, and horseradish peroxidase are consistent with a series of solution biochemical processes with a rate-determining step corresponding to the disproportionation of a luminal–superoxide complex. The effects of the enzymes demonstrate that superoxide is a precursor to the rate-determining intermediate and that both catalase and peroxide enhance a reaction that competes with the rate-limiting process.Key words: chemiluminescence, luminol, amphotericin B, superoxide, HL-60 cells.

scholarly journals Bauhinia forficata link shoot regeneration: histological analysis of organogenesis pathway

2000 ◽  
Vol 43 (4) ◽  
pp. 431-431 ◽  
Author(s):  
Marcia O. Mello ◽  
Murilo Melo ◽  
Beatriz Appezzato-da-Glória
Keyword(s):  
Plant Regeneration ◽  
Culture Medium ◽  
De Novo ◽  
Shoot Elongation ◽  
Small Cells ◽  
Indirect Organogenesis ◽  
Shoot Bud ◽  
Half Strength ◽  
Bauhinia Forficata

Plant regeneration was achieved from cells of callus induced from hypocotyl segments of Bauhinia forficata on half strength Murashige and Skoog culture medium supplemented with several concentrations of BAP. Within 40 days of culture shoot buds formation was observed on callus surface. Calli were then transferred to a same composition culture medium without plant growth regulator in order to induce shoot elongation. Histological studies indicated that in vitro plant regeneration in B. forficata occurred through indirect organogenesis. Meristemoids consisting of small cells with dense cytoplasm and prominent nuclei were randomly distributed throughout the callus surface indicating early stages of shoot bud differentiation. Shoots developed de novo from superficial layers of cells and the pattern of shoot origin and development were very similar to those previously described for other leguminous species.

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