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.

scholarly journals Sperm surface proteins persist after fertilization.

1984 ◽  
Vol 99 (4) ◽  
pp. 1343-1353 ◽  
Author(s):  
G G Gundersen ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Limited Proteolysis ◽  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Gastrula Stage ◽  
Sperm Surface ◽  
Sperm Proteins ◽  
Image Intensification ◽  
Sea Urchin Sperm

Certain sperm components labeled with fluorescein isothiocyanate or its radioactive derivative, 125I-diiodofluorescein isothiocyanate (125IFC), are transferred at fertilization to the egg, where they persist throughout early cleavage stages at a localized site in the embryo cytoplasm (Gabel, C. A., E. M. Eddy, and B. M. Shapiro, 1979, Cell, 18:207-215; Gundersen, G. G., C. A. Gabel, and B. M. Shapiro, 1982, Dev. Biol., 93:59-72). By using image intensification we have extended these observations in the sea urchin to the pluteus larval stage, in which greater than 60% of the embryos have localized fluorescent sperm components. Because of the unusual persistence of the sperm components in the embryo, a characterization of the nature of the labeled species in sea urchin sperm was undertaken. Approximately 10% of the 125IFC was in sperm polypeptides of Mr greater than 15,000. These proteins were on the sperm surface as shown by their sensitivity to externally added proteases. The remainder of the 125IFC in sperm was in several low-molecular-weight species, none of which was 125IFC-derivatized phospholipid. To determine if any labeled sperm polypeptides remained intact in the embryo after fertilization, 125IFC-labeled sperm proteins were recovered from one-cell and late gastrula stage embryos by using an anti-IFC immunoadsorbent. Most of the labeled sperm proteins were degraded shortly after fertilization; however, distinct sets of labeled polypeptides were recovered from both one-cell and gastrula stage embryos. Six of the labeled polypeptides recovered from both embryonic stages had identical SDS gel mobilities as labeled sperm polypeptides. Other polypeptides in the embryos appeared to arise from limited proteolysis of sperm proteins. Thus, in this physiological cell fusion system, individual sperm proteins are transferred to the egg at fertilization, and some persist intact or after specific, limited degradation long after gamete fusion, until at least the late gastrula stage.

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

Selective radiolabeling of cell surface proteins to a high specific activity

Biochemistry ◽  
1987 ◽  
Vol 26 (3) ◽  
pp. 743-750 ◽  
Author(s):  
James A. Thompson ◽  
Alice L. Lau ◽  
Dennis D. Cunningham
Keyword(s):  
Cell Surface ◽  
Specific Activity ◽  
High Specific Activity ◽  
Surface Proteins ◽  
Cell Surface Proteins

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2615-2627 ◽  
Author(s):  
Xiaodong Zhu ◽  
Gregory Mahairas ◽  
Michele Illies ◽  
R. Andrew Cameron ◽  
Eric H. Davidson ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Large Scale ◽  
Surface Proteins ◽  
Matrix Proteins ◽  
Specific Cell ◽  
Gastrula Stage ◽  
Sea Urchin Embryo ◽  
Expressed Sequenced Tags ◽  
Mesenchyme Cells ◽  
Primary Mesenchyme Cells

The primary mesenchyme cells (PMCs) of the sea urchin embryo have been an important model system for the analysis of cell behavior during gastrulation. To gain an improved understanding of the molecular basis of PMC behavior, a set of 8293 expressed sequenced tags (ESTs) was derived from an enriched population of mid-gastrula stage PMCs. These ESTs represented approximately 1200 distinct proteins, or about 15% of the mRNAs expressed by the gastrula stage embryo. 655 proteins were similar (P<10−7 by BLAST comparisons) to other proteins in GenBank, for which some information is available concerning expression and/or function. Another 116 were similar to ESTs identified in other organisms, but not further characterized. We conservatively estimate that sequences encoding at least 435 additional proteins were included in the pool of ESTs that did not yield matches by BLAST analysis. The collection of newly identified proteins includes many candidate regulators of primary mesenchyme morphogenesis, including PMC-specific extracellular matrix proteins, cell surface proteins, spicule matrix proteins and transcription factors. This work provides a basis for linking specific molecular changes to specific cell behaviors during gastrulation. Our analysis has also led to the cloning of several key components of signaling pathways that play crucial roles in early sea urchin development.

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

Abstract We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

scholarly journals Glycoconjugates on the surface of epididymal spermatozoa in a marsupial, the brushtail possum, Trichosurus vulpecula

Reproduction ◽  
2001 ◽  
pp. 165-176 ◽  
Author(s):  
NJ Cooper ◽  
RV McClean ◽  
CM Leigh ◽  
WG Breed
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Western Blots ◽  
Sperm Surface ◽  
Lectin Staining ◽  
Epididymal Spermatozoa ◽  
Cauda Epididymidis ◽  
Caput Epididymidis

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.

CDP-diglyceride hydrolase from pig liver mitochondria

1984 ◽  
Vol 62 (11) ◽  
pp. 1205-1216 ◽  
Author(s):  
D. W. Nicholson ◽  
W. C. McMurray
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
High Specific Activity ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Regulation Of Biosynthesis ◽  
Preparatory Method ◽  
Ph Range ◽  
Triton X

A CDP-diglyceride hydrolase activity, measured by the release of [3H]CMP from labeled CDP-diglyceride, has been identified in pig liver mitochondria. A modified preparatory method for the synthesis of [3H]CDP-diglyceride of high specific activity and purity is also reported. Activity of the hydrolase is enriched 2.5-fold in mitochondrial membranes (over whole mitochondria) and can be solubilized by nonionic detergents such as Triton X-100 with further enrichment of activity (i.e., 7.9-fold). The CDP-diglyceride hydrolase has a Km of 12.8 μM for CDP-diglyceride and a broad pH range with optimum activity at approximately pH 6.2. Of the CDP-diglycerides tested, the hydrolytic rate is highest for dioleoyl CDP-diglyceride. Activity is inhibited by all divalent cations in whole mitochondria, except in the presence of phosphatidylglycerol in which CMP release is stimulated by Co2+ and Mn2+. The increase in CMP release in the presence of Co2+ or Mn2+ can be accounted for entirely by diphosphatidylglycerol synthase activity which requires either cation. This effect is not seen in Triton X-100 solubilized mitochondrial membranes which contain no diphosphatidylglycerol synthase. All preparations are inhibited by mixed phospholipids (Asolectin) and by Trixon X-100 which abolishes activity completely at concentrations greater than 0.5% (w/v). CDP-diglyceride hydrolase is also inhibited by AMP (46%) and by cytidine nucleotides (CTP > CDP > cytidine) except CMP. A role for this activity in the regulation of biosynthesis of mitochondrial polyglycerophosphatides is proposed.

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.

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