scholarly journals Characterization of the antigens recognized by two monoclonal antibodies reactive with basal-layer keratinocytes of human epidermis

1994 ◽  
Vol 299 (3) ◽  
pp. 659-664 ◽  
Author(s):  
G P Roberts
Keyword(s):  
Monoclonal Antibodies ◽  
Basal Layer ◽  
Human Keratinocytes ◽  
Human Epidermis ◽  
Reduced Form ◽  
Lectin Affinity Chromatography ◽  
Major Band ◽  
Reducing Conditions ◽  
Beta 1 Integrin ◽  
Triton X

Two monoclonal antibodies, GR3 and GR4, reactive with the basal-layer keratinocytes of human epidermis, were derived by immunization of Balb/c mice with glycoproteins isolated from cultured keratinocytes by lectin-affinity chromatography. Immunoprecipitation of Triton X-100 extracts from human keratinocytes metabolically labelled with D-[1-14C]glucosamine revealed that GR3 recognized a major glycoprotein with migration properties identical with those of a glycoprotein (reduced form M(r) 126,000) which was previously shown to be implicated in intercellular adhesion [Roberts and Brunt (1985) Biochem J. 232, 67-70]. In their unreduced forms the antigens recognized by GR3 and GR4 both migrated as two bands with M(r) values of 118,000 and 147,000. Comparison of 125I-labelled glycoproteins immunoprecipitated by GR3, GR4 and integrin antibodies revealed that, under reducing conditions, the major band immunoprecipitated by both GR3 and GR4 co-migrated with the alpha 3 and beta 1 integrin chains. In addition the immunoprecipitate obtained with GR4 contained an additional band co-migrating with the alpha 2 integrin chain. Sequential immunoprecipitation studies with GR3, GR4 and integrin antibodies confirmed that GR3 is directed against the alpha 3 integrin chain, whereas GR4 is directed against the beta 1 chain. These studies also indicate that some of the alpha 2 integrin chains on keratinocytes may be associated with a beta-chain not recognized by the antisera against the beta 1 integrin chain used in this study.

Decreased expression of fibronectin and the alpha 5 beta 1 integrin during terminal differentiation of human keratinocytes

1991 ◽  
Vol 98 (2) ◽  
pp. 225-232 ◽  
Author(s):  
L.J. Nicholson ◽  
F.M. Watt
Keyword(s):  
Messenger Rna ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Fibronectin Receptor ◽  
Beta 1 Integrin ◽  
Differentiation Marker ◽  
Unit Gravity Sedimentation

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors

1988 ◽  
Vol 90 (2) ◽  
pp. 201-214 ◽  
Author(s):  
F. Grinnell ◽  
C.H. Ho ◽  
T.L. Tuan
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Binding Sites ◽  
Cell Spreading ◽  
The Other ◽  
Immunofluorescence Staining ◽  
Baby Hamster Kidney ◽  
Bhk Cells ◽  
Reducing Conditions ◽  
Latex Beads

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHK1 staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHK1 and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their ‘capping’ response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5- to 20-fold more efficiently than anti-BHK1 beads. The binding sites for anti-BHK1 and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHK1 binding sites contained 50K (K = 10(3) Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.

scholarly journals The hyaluronate synthase from a eukaryotic cell line

1993 ◽  
Vol 290 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L Klewes ◽  
E A Turley ◽  
P Prehm
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Affinity Chromatography ◽  
Cell Line ◽  
Aqueous Phase ◽  
Plasma Membranes ◽  
Eukaryotic Cell ◽  
Molecular Masses ◽  
Triton X ◽  
Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

scholarly journals Towards Initial Indications for a Thiol-Based Redox Control of Arabidopsis 5-Aminolevulinic Acid Dehydratase

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 152 ◽  
Author(s):  
Daniel Wittmann ◽  
Sigri Kløve ◽  
Peng Wang ◽  
Bernhard Grimm
Keyword(s):  
Aminolevulinic Acid ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Reduced Form ◽  
Redox Control ◽  
In Planta ◽  
Acid Dehydratase ◽  
Reducing Conditions ◽  
Alad Activity

Thiol-based redox control is one of the important posttranslational mechanisms of the tetrapyrrole biosynthesis pathway. Many enzymes of the pathway have been shown to interact with thioredoxin (TRX) and Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase C (NTRC). We examined the redox-dependency of 5-aminolevulinic acid dehydratase (ALAD), which catalyzed the conjugation of two 5-aminolevulinic acid (ALA) molecules to porphobilinogen. ALAD interacted with TRX f, TRX m and NTRC in chloroplasts. Consequently, less ALAD protein accumulated in the trx f1, ntrc and trx f1/ntrc mutants compared to wild-type control resulting in decreased ALAD activity. In a polyacrylamide gel under non-reducing conditions, ALAD monomers turned out to be present in reduced and two oxidized forms. The reduced and oxidized forms of ALAD differed in their catalytic activity. The addition of TRX stimulated ALAD activity. From our results it was concluded that (i) deficiency of the reducing power mainly affected the in planta stability of ALAD; and (ii) the reduced form of ALAD displayed increased enzymatic activity.

Activation of human keratinocyte migration on type I collagen and fibronectin

1990 ◽  
Vol 96 (2) ◽  
pp. 197-205
Author(s):  
M. Guo ◽  
K. Toda ◽  
F. Grinnell
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Type I ◽  
Integrin Subunit ◽  
Basal Cells ◽  
Keratinocyte Migration ◽  
Beta 1 Integrin ◽  
Cells Cultured

The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by keratinocytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained beta 1 integrin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, beta 1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In keratinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of beta 1 integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, beta 1 integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of beta 1 integrin subunits accompany development of migratory competence.

scholarly journals 272 Understanding cell heterogeneity in the basal layer of human epidermis

2019 ◽  
Vol 139 (9) ◽  
pp. S261
Author(s):  
D. Belokhvostova ◽  
C. Philippeos ◽  
L. Mamanova ◽  
C. Smith ◽  
M. Haniffa ◽  
...  
Keyword(s):  
Basal Layer ◽  
Human Epidermis ◽  
Cell Heterogeneity

scholarly journals The Epidermal Stem Cell Compartment: Variation in Expression Levels of E–Cadherin and Catenins Within the Basal Layer of Human Epidermis

1997 ◽  
Vol 45 (6) ◽  
pp. 867-874 ◽  
Author(s):  
Jean-Pierre Molès ◽  
Fiona M. Watt
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Basal Layer ◽  
Human Epidermis ◽  
Proliferative Potential ◽  
Stem Cell Markers ◽  
Cell Compartment ◽  
Stem Cell Compartment ◽  
E Cadherin ◽  
Cell Cell

The basal layer of the epidermis contains two types of proliferating keratinocyte: stem cells, with high proliferative potential, and transit amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. It has been shown previously that two- to three-fold differences in the average staining intensity of fluorescein-conjugated antibodies to β1 integrin subunits reflect profound differences in the proliferative potential of keratinocytes, with integrin-bright populations being enriched for stem cells. In the search for additional stem cell markers, we have stained sections of normal human epidermis with antibodies to proteins involved in intercellular adhesion and quantitated the fluorescence of individual cell-cell borders. In the basal layer, patches of brightly labeled cells were detected with antibodies to E-cadherin, β-catenin, and γ-catenin, but not with antibodies to P-cadherin, α-catenin, or with pan-desmocollin and pan-desmoglein antibodies. In the body sites examined, palm and foreskin, integrinbright regions were strongly labeled for γ-catenin and weakly labeled for E-cadherin and β-catenin. Our data suggest that there are gradients of both cell-cell and cell-extracellular matrix adhesiveness within the epidermal basal layer and that the levels of E-cadherin and of β-and γ-catenin may provide markers for the stem cell compartment, stem cells expressing relatively higher levels of γ-catenin and lower levels of E-cadherin and β-catenin than other basal keratinocytes.

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