The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

scholarly journals Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

2007 ◽  
Vol 405 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Jørgen de Jonge ◽  
Johanna M. Leenhouts ◽  
Marijke Holtrop ◽  
Pieter Schoen ◽  
Peter Scherrer ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Gradient Centrifugation ◽  
Cationic Lipid ◽  
Sucrose Density Gradient ◽  
Target Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Viral Membrane ◽  
Viral Envelopes

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

scholarly journals Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1970 ◽  
Vol 119 (4) ◽  
pp. 773-784 ◽  
Author(s):  
J. A. Smith ◽  
L. Martin ◽  
R. J. B. King ◽  
M. Vértes
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Nuclear Protein ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Nuclear Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Mouse Uterus

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17β given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [14C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17β has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [14C]lysine into stromal nuclear proteins, but changes after oestradiol-17β treatment were similar to those seen in epithelium with oestradiol-17β alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17β. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17β alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-3H]oestradiol-17β by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.

PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

1969 ◽  
Vol 62 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Olav Unhjem ◽  
Kjell J. Tveter ◽  
Asbjørn Aakvaag
Keyword(s):  
Proteolytic Enzymes ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Macromolecular Complex ◽  
Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

CHARACTERIZATION OF THE SPECIFIC ANDROGEN RECEPTORS IN THE HUMAN PROSTATE GLAND

1973 ◽  
Vol 57 (3) ◽  
pp. 371-384 ◽  
Author(s):  
W. I. P. MAINWARING ◽  
E. J. G. MILROY
Keyword(s):  
Binding Proteins ◽  
Androgen Receptors ◽  
Gradient Centrifugation ◽  
Prostate Gland ◽  
Sucrose Density Gradient ◽  
Human Prostate ◽  
Sucrose Density Gradient Centrifugation ◽  
Androgen Binding

SUMMARY A search has been conducted for specific androgen-binding proteins in soluble extracts of normal human prostate tissue and in surgical samples removed at retropubic prostatectomy for benign prostatic hyperplasia. Proteins with a particularly high affinity for a metabolite of testosterone, 5α-dihydrotestosterone, have been identified in studies on the binding of 3H-labelled steroids in vitro. The 5α-dihydrotestosterone-protein complexes have been partially characterized using sucrose density gradient centrifugation and gel-exclusion chromatography. The androgen receptors in the human prostate gland are remarkably similar to those previously described in the accessory sexual glands of experimental animals. These findings have been confirmed by the analysis of extracts of hyperplastic prostate glands labelled by the administration of [3H]testosterone in vivo. No attempt was made to correlate the degree of binding with the histological appearance of the specimens of hyperplastic prostate or to determine whether the receptors were principally present in the hyperplastic nodules. Such androgen-binding proteins were not present in serum, skeletal muscle or adipose tissue. The possible relevance of these findings to the onset of clinical disorders in the human prostate gland is briefly discussed.

scholarly journals Intestine epithelial cell-derived extracellular vesicles alleviate inflammation induced by Clostridioides difficile TcdB through the activity of TGF-β1

2021 ◽  
Author(s):  
Shuangshuang Wan ◽  
Guangzhong Song ◽  
Hui Hu ◽  
Yaqing Xu ◽  
Peng Zeng ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Extracellular Vesicles ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Extracellular Vesicle ◽  
Sucrose Density Gradient Centrifugation ◽  
Clostridioides Difficile ◽  
Intestinal Immune System

Abstract Objective: Clostridioides difficile infection (CDI) has been primarily associated with the toxin B (TcdB), which can activate the intestinal immune system and lead to pathological damage. Even though the biological functions of intestine epithelial cell-derived extracellular vesicles (I-Evs) have been well documented, the role of I-Evs in the process of CDI is still unknown. Methods: I-Evs were isolated from mouse intestine tissues by ultracentrifugation protocol, identified by electron microscopy, nanoparticle tracking, sucrose density gradient centrifugation, and western blotting. Intestinal pathological damage was measured after intraperitoneal injection of TcdB into mice. Results: We isolated I-Evs ranging from 100–200 nm in mean diameter, with a density of 1.09-1.17 g/mL. These I-Evs expressed the extracellular vesicle-associated specific surface markers, CD63 and TSG101. In vitro, 50 µg I-Evs decreased the expression of IL-6, TNF-a, IL-1β, and IL-22 induced by 0.8 ng/mL C. difficile TcdB, and increased expression of TGF-b1. In vivo, I-Evs also promoted regulatory T cell induction, which improved the survival rate of mice up to 80% relative to C. difficile TcdB mice, dependent on the TGF-b1 signalling pathway. Conclusion: As an emerging immunotherapy, I-Evs can reduce the intraperitoneal infection induced by C. difficile TcdB and improve survival in mice.

scholarly journals Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1971 ◽  
Vol 123 (4) ◽  
pp. 635-641 ◽  
Author(s):  
R. M. E. Parkhouse
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Immunoglobulin M ◽  
Sucrose Density Gradient Centrifugation ◽  
Chain Synthesis

Immunoglobulin M (IgM) biosynthesis was studied with mouse plasma-cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with [3H]leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The tumour secretes IgM and light chains. ‘Pulse–chase’ experiments indicated average secretion times of 1.5h for light chain and 2.5h for IgM. The order of disulphide-bond assembly within the cell was shown to be heavy chain+light chain → heavy chain–light chain intermediate → IgMs. The 7S subunit (IgMs) was polymerized into IgM just before or at the time of secretion. Measurements of heavy-chain/light-chain radioactivity ratios in intracellular HL and IgMs and secreted IgM demonstrated the existence of a light-chain pool participating in IgM biosynthesis. The size of the light-chain pool, together with analysis of clones isolated in vivo, suggested that the tumour contains cells in which light-chain synthesis is in excess of heavy-chain production.

V. EFFECTS OF THE ANTI-ANDROGEN TSAA-291 ON THE ANDROGEN-RECEPTOR COMPLEX FORMATION FROM [3H]TESTOSTERONE IN RAT VENTRAL PROSTATES

1979 ◽  
Vol 92 (3_Supplb) ◽  
pp. S67-S81 ◽  
Author(s):  
Katsuichiro Sudo ◽  
Keiji Yoshida ◽  
Yoshiaki Kimura ◽  
Ryo Nakayama
Keyword(s):  
Androgen Receptor ◽  
Gradient Centrifugation ◽  
Control Level ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Sucrose Density Gradient Centrifugation ◽  
Macromolecular Complexes

ABSTRACT Intramuscular administration of a new steroidal anti-androgen, TSAA-291(16β-ethyl-17β-hydroxy-4-oestren-3-one), in doses of 0.05, 0.5 and 5 mg/kg body weight reduced the in vivo uptake of [3H]testosterone by the ventral prostate of castrated rats to 78, 59 and 37% of the control level, respectively. Analysis on subcellular fractions of the prostate by gel-filtration and sucrose density-gradient centrifugation followed by thin layer chromatographic identification of testosterone metabolites revealed that 5α-dihydrotestosterone(5α-DHT) which was found to be largely bound to macromolecules in the cytosol and nucleus was the predominant metabolite even in the presence of the anti-androgen, and radioactivities corresponding to the 5α-DHT-macromolecular complexes were decreased by the anti-androgen. TSAA-291 also inhibited the in vitro formation of the 5α-DHT-macromolecular complexes in both cytosol and nucleus from minced prostates incubated with [3H]testosterone. The importance of the findings is discussed in connection with the mode of anti-androgenic action of TSAA-291 in terms of the interaction with the androgen receptor.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

scholarly journals Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).

1988 ◽  
Vol 107 (5) ◽  
pp. 1767-1776 ◽  
Author(s):  
M D Neely ◽  
K Boekelheide
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sertoli Cell ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytoplasmic Dynein ◽  
Microtubule Assembly ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Stage ◽  
Structural And Functional Properties

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.

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