scholarly journals Dye- and fluorescence-based assay to characterize symplastic and apoplastic trafficking in soybean (Glycime max L.) endosperm

2019 ◽  
Vol 60 (1) ◽  
Author(s):  
Ming-Der Shih ◽  
Jian-Shin Lin ◽  
Mei-Jane Fang ◽  
Yuan-Ching Tsai ◽  
Yue-Ie C. Hsing
Keyword(s):  
Fusion Proteins ◽  
Sperm Nucleus ◽  
Size Exclusion ◽  
Simple Diffusion ◽  
Symplastic Transport ◽  
Legume Crop ◽  
Embryo Cells ◽  
Well Differentiated ◽  
Cotyledon Cells ◽  
Zigzag Shape

Abstract Background Endosperm is a triploid tissue in seed resulting from a sperm nucleus fused with the binucleate central cell after double fertilization. Endosperm may be involved in metabolite production, solute transport, nutrient storage, and germination. In the legume family (Fabaceae), with the greatest number of domesticated crops, approximately 60% of genera contain well-differentiated endosperm in mature seeds. Soybean seeds, the most important legume crop in the worlds, have endosperm surrounding embryos during all stages of seed development. However, the function of soybean endosperm is still unknown. Results Flow cytometry assay confirmed that soybean endosperm was triploid. Cytobiological observation showed that soybean endosperm cells were alive with zigzag-shape cell wall. Soybean endosperm cells allowed fusion proteins (42 kDa) to move from bombarded cells to adjacent unbombarded-cells. Such movement is not simple diffusion because the fusion proteins failed to move into dead cells. We used symplastic tracers to test the transport potential of soybean endosperm. Small organic dye and low-molecular-weight symplastic tracers revealed fast symplastic transport. After a treatment of an inhibitor of ATPase, N,N′-dicyclohexylcarbodiimide (DCCD), symplastic transport was blocked, but all tracers still showed fast apolopastic transport. The transport speed of 8-hydroxypyrene-1,3,6-trisulfonic acid in endosperm was 1.5 to 3 times faster than in cotyledon cells or Arabidopsis embryos. Conclusions Soybean endosperm is a membrane-like, semi-transparent, and fully active tissue located between the seed coat and cotyledon. Soybean endosperm cells allowed macromolecules to move fast via plasmodesmata transport. The size exclusion limit is larger for soybean endosperm cells than its cotyledon or even Arabidopsis embryo cells. Soybean endosperm may be involved in fast and horizontal transport during the mid-developmental stage of seeds.

Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate

1993 ◽  
Vol 13 (12) ◽  
pp. 7892-7900
Author(s):  
D C Flynn ◽  
T H Leu ◽  
A B Reynolds ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Actin Filament ◽  
Chicken Embryo ◽  
Fusion Proteins ◽  
Structural Integrity ◽  
Sequence Data ◽  
Stable Complex ◽  
Sh3 Domains ◽  
Cell Extracts ◽  
Embryo Cells

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.

Bispecific Fynomer-antibody fusion proteins targeting two epitopes on HER2.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2575-2575
Author(s):  
Frederic Mourlane ◽  
Isabella Toller ◽  
Simon Brack ◽  
Dragan Grabulovski ◽  
Julian Bertschinger-Ehrler
Keyword(s):  
Fusion Proteins ◽  
Chemical Properties ◽  
Size Exclusion ◽  
Tumor Xenograft ◽  
Antitumoral Activity ◽  
Phage Display Technology ◽  
Genetic Fusion ◽  
High Yields

2575 Background: Fynomers are small binding proteins (7 kDa) derived from the SH3 domain of Fyn kinase. They can be engineered to yield specific and high-affinity binding domains to target proteins of interest. In the past, we have isolated Fynomers with excellent physico-chemical properties binding to 16 different antigens. One important application of the Fynomer technology represents the genetic fusion of a Fynomer to an antibody to provide bispecific fusion proteins. Methods: Using phage display technology we have isolated Fynomers binding to tumor-associated antigens. After genetic fusion of these Fynomers to antibodies of interest the resulting bispecific proteins were evaluated in vitro and in vivo for their antitumoral activity. Results: The fusion of Fynomers to the antibodies of interest did not alter the favorable biophysical properties of the antibodies. First, the Fynomer-antibody fusion proteins could be purified with the same high yields from the supernatant of transiently transfected CHO cells as the unmodified antibodies (in the range of 100 mg/L). Second, the purified fusion proteins were monomeric and showed no signs of aggregation even after four months of storage at 4 °C or -20 °C in PBS as determined by size exclusion chromatography. Third, the Fc-mediated effector functions of antibodies were not altered by their fusion with Fynomers. Antibody-dependent cellular cytotoxicity (ADCC) was maintained, and the affinity to the neonatal Fc-receptor (FcRn) was similar as observed for the unmodified antibody. In addition, the bispecific Fynomer-antibody fusion proteins demonstrated excellent growth inhibition of tumor cells in vitro and high efficacy in vivo in tumor xenograft mouse models. Conclusions: These encouraging preclinical results indicate that the bispecific Fynomer-antibody fusions have highly promising properties with a great potential for further preclinical and clinical development.

scholarly journals Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate.

1993 ◽  
Vol 13 (12) ◽  
pp. 7892-7900 ◽  
Author(s):  
D C Flynn ◽  
T H Leu ◽  
A B Reynolds ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Actin Filament ◽  
Chicken Embryo ◽  
Fusion Proteins ◽  
Structural Integrity ◽  
Sequence Data ◽  
Stable Complex ◽  
Sh3 Domains ◽  
Cell Extracts ◽  
Embryo Cells

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.

scholarly journals Selenium, selenoproteins and selenometabolites in mothers and babies at the time of birth

2017 ◽  
Vol 117 (9) ◽  
pp. 1304-1311 ◽  
Author(s):  
Cristina Santos ◽  
Eduardo García-Fuentes ◽  
Belén Callejón-Leblic ◽  
Tamara García-Barrera ◽  
José Luis Gómez-Ariza ◽  
...  
Keyword(s):  
Gestational Age ◽  
Cross Sectional Study ◽  
Maternal Serum ◽  
Size Exclusion ◽  
Total Serum ◽  
Cross Sectional ◽  
Simple Diffusion ◽  
Cord Serum ◽  
In Series ◽  
Adverse Pregnancy

AbstractThe deficiency of Se, an essential micronutrient, has been implicated in adverse pregnancy outcomes. Our study was designed to determine total serum Se, selenoproteins (extracellular glutathione peroxidase (GPx-3), selenoprotein P (SeP)), selenoalbumin (SeAlb) and selenometabolites in healthy women and their newborns at delivery. This cross-sectional study included eighty-three healthy mother–baby couples. Total Se and Se species concentrations were measured in maternal and umbilical cord sera by an in-series coupling of two-dimensional size-exclusion and affinity HPLC. Additional measurements of serum SeP concentration and of serum GPx-3 enzyme activity were carried out using ELISA. Total Se concentration was significantly higher in maternal serum than in cord serum (68·9 (sd 15·2) and 56·1 (sd 14·6) µg/l, respectively; P<0·01). There were significant correlations between selenoprotein and SeAlb concentrations in mothers and newborns, although they also showed significant differences in GPx-3 (11·2 (sd 3·7) v. 10·5 (sd 3·5) µg/l; P<0·01), SeP (42·5 (sd 9·5) v. 28·1 (sd 7·7) µg/l; P<0·01) and SeAlb (11·6 (sd 3·6) v. 14·1 (sd 4·3) µg/l; P<0·01) concentrations in maternal and cord sera, respectively. Serum GPx-3 activity and concentration were positively correlated in mothers (r 0·33; P=0·038) but not in newborns. GPx-3 activity in cord serum was significantly correlated with gestational age (r 0·44; P=0·009). SeAlb concentration was significantly higher in babies, whereas SeP and GPx-3 concentrations were significantly higher in mothers. The differences cannot be explained by simple diffusion; specific transfer mechanisms are probably involved. GPx-3 concentrations in mothers, at delivery, are related to maternal Se status, whereas the GPx-3 activity in cord serum depends on gestational age.

Ultrastructure of NPC/HK1 cells heterotransplanted in athymic nude mice

1982 ◽  
Vol 40 ◽  
pp. 236-237
Author(s):  
E.C. Chew ◽  
C.L. Li ◽  
D.P. Huang ◽  
H.C. Ho ◽  
L.S. Mak ◽  
...  
Keyword(s):  
Cell Line ◽  
Nude Mice ◽  
Biopsy Specimen ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Recurrent Tumour ◽  
Athymic Nude Mice ◽  
Cell Line Establishment ◽  
Fine Structures ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been established from a biopsy specimen of a recurrent tumour of the nasopharynx which was histologically diagnosed as a moderately to well differentiated squamous cell carcinoma. A definite decrease in the amount of tonofilaments and desmosomes in the NPC/HK1 cells during the cell line establishment was observed. The present communication reports on the fine structures of the NPC/HK1 cells heterotraneplanted in athymic nude mice.

An Established Epithelial Cell Line (NPC/HK1) from a Nasopharyngeal Carcinoma - II. A Sem Study

1982 ◽  
Vol 40 ◽  
pp. 224-225
Author(s):  
Li C.L. ◽  
Chew E.C. ◽  
Huang D.P. ◽  
Ho H.C. ◽  
Mak L.S. ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Epithelial Cell ◽  
Surface Morphology ◽  
Cell Line ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Cells In Culture ◽  
Sem Study ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been successfully established from a nasopharyngeal carcinoma of the moderately to well differentiated squamous type. The present communication reports on the surface morphology of the NPC/HK1 cells in culture.

Morphological Examination of a Herpes-type Virus Indigenous for Tree Shrews

1970 ◽  
Vol 28 ◽  
pp. 160-161
Author(s):  
J. P. Brunschwig ◽  
R. M. McCombs ◽  
R. Mirkovic ◽  
M. Benyesh-Melnick
Keyword(s):  
Electron Microscopy ◽  
Soft Tissue ◽  
Chick Embryo ◽  
Soft Tissue Sarcoma ◽  
Cell Cultures ◽  
Tree Shrew ◽  
Type Virus ◽  
Morphological Examination ◽  
Tree Shrews ◽  
Embryo Cells

A new virus, established as a member of the herpesvirus group by electron microscopy, was isolated from spontaneously degenerating cell cultures derived from the kidneys and lungs of two normal tree shrews. The virus was found to replicate best in cells derived from the homologous species. The cells used were a tree shrew cell line, T-23, which was derived from a spontaneous soft tissue sarcoma. The virus did not multiply or did so poorly for a limited number of passages in human, monkey, rodent, rabbit or chick embryo cells. In the T-23 cells, the virus behaved as members of the subgroup B of herpesvirus, in that the virus remained primarily cell associated.

Spontaneous Production of Type C Virus Particles in a Culture Derived from Rat Embryo Cells

1972 ◽  
Vol 30 ◽  
pp. 288-289
Author(s):  
Elizabeth S. Priori ◽  
T. Shigematsu ◽  
B. Myers ◽  
L. Dmochowski
Keyword(s):  
Virus Particle ◽  
Mouse Embryo ◽  
Calf Serum ◽  
Embryo Cell ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Mouse Embryo Cell ◽  
Mature Virus Particle ◽  
Embryo Cells ◽  
Type C

Spontaneous release of type C virus particles in long-term cultures of mouse embryo cells as well as induction of similar particles in mouse embryo cell cultures with IUDR or BUDR have been reported. The presence of type C virus particles in cultures of normal rat embryos has not been reported.NB-1, a culture derived from embryos of a New Zealand Black (NB) rat (rats obtained from Mr. Samuel M. Poiley, N.C.I., Bethesda, Md.) and grown in McCoy's 5A medium supplemented with 20% fetal calf serum was passaged weekly. Extracellular virus particles similar to murine leukemia particles appeared in the 22nd subculture. General appearance of cells in passage 23 is shown in Fig. 1. Two budding figures and one immature type C virus particle may be seen in Fig. 2. The virus particles and budding were present in all further passages examined (currently passage 39). Various stages of budding are shown in Figs. 3a,b,c,d. Appearance of a mature virus particle is shown in Fig. 4.

A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

1975 ◽  
Vol 33 ◽  
pp. 594-595
Author(s):  
A. Sosa ◽  
L. Calzada
Keyword(s):  
High Resolution ◽  
Atpase Activity ◽  
Nuclear Membrane ◽  
Human Sperm ◽  
Sperm Nucleus ◽  
Human Spermatozoa ◽  
Cytochemical Study ◽  
Membrane Bound ◽  
Sperm Nuclei ◽  
Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

Motility of leukemic lymphocytes

1990 ◽  
Vol 48 (3) ◽  
pp. 368-369
Author(s):  
Manoj Raje ◽  
Karvita B. Ahluwalia
Keyword(s):  
Quantitative Data ◽  
Laser Doppler Velocimetry ◽  
Acute Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Cellular Space ◽  
Differentiated Cells ◽  
Poorly Differentiated ◽  
Solid Tissues ◽  
Well Differentiated ◽  
Reduced Mobility

In Acute Lymphocytic Leukemia motility of lymphocytes is associated with dissemination of malignancy and establishment of metastatic foci. Normal and leukemic lymphocytes in circulation reach solid tissues where due to in adequate perfusion some cells get trapped among tissue spaces. Although normal lymphocytes reenter into circulation leukemic lymphocytes are thought to remain entrapped owing to reduced mobility and form secondary metastasis. Cell surface, transmembrane interactions, cytoskeleton and level of cell differentiation are implicated in lymphocyte mobility. An attempt has been made to correlate ultrastructural information with quantitative data obtained by Laser Doppler Velocimetry (LDV). TEM of normal & leukemic lymphocytes revealed heterogeneity in cell populations ranging from well differentiated (Fig. 1) to poorly differentiated cells (Fig. 2). Unlike other cells, surface extensions in differentiated lymphocytes appear to originate by extrusion of large vesicles in to extra cellular space (Fig. 3). This results in persistent unevenness on lymphocyte surface which occurs due to a phenomenon different from that producing surface extensions in other cells.

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