scholarly journals Immunochemical characterization of l-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues

1995 ◽  
Vol 309 (3) ◽  
pp. 993-998 ◽  
Author(s):  
D Boivin ◽  
D Bilodeau ◽  
R Béliveau
Keyword(s):  
Polyclonal Antibodies ◽  
Protein A ◽  
Cell Types ◽  
Critical Micellar Concentration ◽  
Kidney Cortex ◽  
Rat Tissues ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Protein Carboxyl Methyltransferase ◽  
Mammalian Tissues

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

scholarly journals Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.

1996 ◽  
Vol 44 (9) ◽  
pp. 1013-1019 ◽  
Author(s):  
K Zaar
Keyword(s):  
Protein A ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Systematic Investigation ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Bovine Kidney

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

scholarly journals Immunoelectron microscopic localization of the isozymes of L-alpha-hydroxyacid oxidase in renal peroxisomes of beef and sheep: evidence of distinct intraorganellar subcompartmentation.

1991 ◽  
Vol 39 (6) ◽  
pp. 801-808 ◽  
Author(s):  
K Zaar ◽  
H D Fahimi
Keyword(s):  
Protein A ◽  
Kidney Cortex ◽  
Rat Tissues ◽  
Marker Enzyme ◽  
Peroxisomal Membrane ◽  
Thin Sections ◽  
The Matrix ◽  
Liver And Kidney ◽  
Marginal Plates

L-alpha-hydroxyacid oxidase (HAOX), a peroxisomal marker enzyme in mammals, exists in two isozymic forms, HAOX A (EC 1.1.3.1) and HAOX B (EC 1.3.4.2), which differ in their substrate specificity. In rat tissues HAOX A is found exclusively in hepatocyte peroxisomes and HAOX B in renal peroxisomes. Recently we found enzymatic evidence that highly purified peroxisome preparations from beef and sheep kidney cortex contain both isozymes. In situ, the peroxisomes in the proximal tubule cells of both species exhibit peculiar angular outlines apparently due to the presence of multiple marginal plates. Marginal plates are plate-like crystalline matrix inclusions which are apposed to the inner aspect of the peroxisomal membrane. In this study monospecific antibodies against HAOX A and B proteins purified from rat liver and kidney, respectively, were raised in rabbits and used to study the intraorganellar localization of each isozyme in beef and sheep kidney cortex peroxisomes. Incubation of ultra-thin sections of LR White-embedded tissue with anti-HAOX A or B followed by protein A-gold revealed that in both species HAOX A is present diffusely in the peroxisomal matrix, whereas HAOX B is localized almost exclusively in the membrane associated marginal plates. This is the first report on the in situ immunocytochemical characterization of marginal plates, which are the most common inclusions in the matrix of renal peroxisomes.

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor

1990 ◽  
Vol 126 (1) ◽  
pp. 17-NP ◽  
Author(s):  
H. Takeda ◽  
G. Chodak ◽  
S. Mutchnik ◽  
T. Nakamoto ◽  
C. Chang
Keyword(s):  
Androgen Receptor ◽  
Polyclonal Antibodies ◽  
Cell Types ◽  
Reproductive Organs ◽  
Specific Cell ◽  
Rat Tissues ◽  
Positive Staining ◽  
Nuclear Staining ◽  
Mouse Tissues ◽  
Human And Mouse

ABSTRACT Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein. All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues. Journal of Endocrinology (1990) 126, 17–25

Role of PKC isozymes in water transport in toad urinary bladder

1991 ◽  
Vol 49 ◽  
pp. 302-303
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Protein A ◽  
Cell Types ◽  
Leukemic Cells ◽  
Toad Urinary Bladder ◽  
Pkc Isozymes ◽  
Antibody Labeling

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

1990 ◽  
Vol 48 (3) ◽  
pp. 944-945
Author(s):  
Ichiro Yamamoto ◽  
Toshiaki Tachibana ◽  
Hiroko Maruyama ◽  
Noriyuki Komatsu ◽  
Hiroyuki Kuramoto ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Cell Lines ◽  
Endometrial Adenocarcinoma ◽  
Protein A ◽  
Rabbit Antiserum ◽  
Carcinoma Cells ◽  
Affinity Column ◽  
Antibody Technique ◽  
Galactosyl Transferase ◽  
C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

scholarly journals Development, characterization, and applications of multi-material stereolithography bioprinting

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bagrat Grigoryan ◽  
Daniel W. Sazer ◽  
Amanda Avila ◽  
Jacob L. Albritton ◽  
Aparna Padhye ◽  
...  
Keyword(s):  
Material Selection ◽  
Cell Types ◽  
Fluorescent Tracers ◽  
Regional Feature ◽  
Multicellular Aggregates ◽  
Distinct Cell ◽  
Adenocarcinoma Cells ◽  
Mammalian Tissues ◽  
Heterotypic Interactions ◽  
Feature Alignment

AbstractAs a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure–function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-β1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments.

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