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Biomedical subjects

I A Bernstein

Publications and source records attributed to I A Bernstein.

At least 37 records · Page 2Linked to original sources
PubMed1988

Macromolecular metabolism of a differentiated rat keratinocyte culture system following exposure to sulfur mustard.

A method for producing a stratified, squamous epithelium in vitro by cultivating rat keratinocytes on nylon membranes has been developed in this laboratory. This epidermal-like culture is being used to obtain a better understanding of the mechanism of skin vesication after topical exposure to the sulfur mustard bis(beta-chloroethyl) sulfide (BCES) dissolved in a selected solvent. Radiolabeled macromolecular precursors (thymidine, uridine, and leucine) have been used to study the effect of BCES on the synthesis of DNA, RNA, and protein, respectively, after topical exposure to the mustard at concentrations of 0.01-500 nmol/cm2 dissolved in 70% dimethyl sulfoxide (DMSO). From these and other studies it has been determined that exposure to even the low concentration of 0.01 nmol BCES/cm2 for 30 min results in significant inhibition of [3H]thymidine incorporation, although complete recovery occurs by 24 h. Significant inhibition of [3H]uridine and [14C]leucine incorporation is observed only after exposure to much higher concentrations of BCES (10-500 nmol/cm2). This suggests a very early lesion in macromolecular metabolism with DNA being the primary target.

Animals↗
PubMed1987

12-O-tetradecanoylphorbol-13-acetate induces an immediate, but transient increase in mitotic activity uncoupled from DNA synthesis in the monolayer cultures of rat keratinocyte.

A single wave of mitotic activity was observed in a monolayer culture of rat keratinocytes immediately after exposure to 12-O-tetradecanoylphorbol-13-acetate. A peak for cells in prophase, observed at 10 min after the exposure, was followed by a peak for metaphase at 20 min, for anaphase at 25 min and telophase at 30 min after the exposure. Thereafter, the mitotic activity began to subside. This transient stimulation of mitotic activity resulted in an increase of population density in the monolayer culture. There was neither a stimulation of DNA synthesis during this period nor a change of the DNA content after the mitotic activity was completed. This single burst of synchronous mitotic activity which did not require a substantial stimulation of DNA synthesis suggests that the effect was on the initiation process of mitosis among a subpopulation of cells, presumably cells delayed in the G2 phase of the cell cycle.

Animals↗
PubMed1987

Exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) induces the synthesis of histidine-rich protein (filaggrin) in monolayer cultures of rat keratinocytes.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, on epidermal differentiation has been studied by investigating the appearance of the epidermal differentiative marker protein, histidine-rich protein (HRP), in monolayer cultures of rat keratinocytes grown in low-calcium (0.1 mM) medium. Monoclonal antibody, 3F6-6, raised against the 60K HRP (HRPII) derived from the epidermis of the newborn rat was utilized to detect HRP in cultured keratinocytes. As a major component of keratohyalin in the cells of the granular layer in epidermis, HRP is not normally found in a monolayer culture of rat keratinocytes containing a high population of proliferating cells. However, when these cultures were exposed to 10 ng/ml of TPA, DNA and protein synthesis were decreased by up to 95% and 60%, respectively, and the cells in the monolayer culture became HRP-positive within 18-24 h. TPA has been shown to cause a withdrawal of the basal cells from the cell cycle and a commitment of these cells to an accelerated rate of terminal differentiation. Exposure to TPA at the same time that the calcium level of the monolayer cultures was raised to 2.0 mM in order to allow stratification resulted in positive immunostaining for HRP much earlier and much more intensively than in the absence of TPA. TPA also enhanced stratification of these cultures; however, the positive expression of HRP preceded the stratification. These observations support the view that the induction of terminal differentiation occurs independently of stratification and that the process of terminal differentiation (maturation) of basal cells precedes and triggers the movement of these cells to a suprabasal position (stratification).

Animals↗
PubMed1987

Changes in lectin binding by differentiating cutaneous keratinocytes from the newborn rat.

Surface glycoconjugates of cells from the basal layer of the skin of the newborn rat bind the isolectin I-B4 from Griffonia simplicifolia (GS I-B4) (alpha-D-galactosyl specificity). Surface glycoconjugates of the differentiated cells from the spinous and lower granular layers bind Ulex europeus agglutinin I (UEA) (alpha-L-fucosyl specificity). The change from GS I-B4 binding to UEA binding was studied in rat keratinocytes that were cultured as a monolayer in low-calcium medium until confluence, and then induced to stratify and terminally differentiate by raising the calcium concentration of the medium. The cells in the monolayer had basal cell morphology and exhibited surface binding of GS I-B4. However, at confluence, 30-40% of these cells also showed surface binding of UEA. There was an increase with time in the number of cells which bound both GS I-B4 and UEA. Raising the calcium concentration of the medium resulted in an increase in UEA binding. Cells of the upper layers of the stratifying cultures showed intense UEA binding but did not show any GS I-B4 binding. Double staining of frozen sections of newborn rat skin with fluorescein-conjugated GS I-B4 and rhodamine-conjugated UEA revealed that the surfaces of cells from the lower spinous layer bound both lectins. Thirty percent of the major glycoprotein fraction, that was isolated from the membranes of the epidermal cells of the newborn rat and was bound to an affinity column of UEA-Sepharose 4B, was also bound to an affinity column of GS I-B4-Sepharose 4B. These results indicate that surface glycoconjugates of rat keratinocytes differentiating in culture exhibit a change from GS I-B4 binding to UEA binding; the change in the cell surface glycoconjugates that results in the appearance of UEA binding, a feature of differentiated cells, occurs independently of stratification; and the change from GS I-B4 binding to UEA binding probably involves an "intermediate" glycoconjugate that binds both GS I-B4 and UEA and is found on the surface of cells from the lower spinous layer of the epidermis of the newborn rat.

Animals↗
PubMed1987

Relationship between cell differentiation and binding of fluorescein isothiocyanate (FITC)-conjugated insulin of keratinocytes in culture.

Basal-type keratinocytes, isolated from newborn rat skin and separated on Percoll density gradients, proliferate in low (0.1 mM) calcium medium and, after raising the calcium level to normal (1.96 mM), stratify. Cells in the low calcium culture do not have extensive cell-cell connections, as seen with fluorescein isothiocyanate (FITC)-conjugated insulin. Fluorescein isothiocyanate-conjugated concanavalin A and Griffonia simplicifolia isolectin B4, but not peanut agglutinin (PNA), fluorescently label these cells. In 3-day-old low calcium cultures, within 2 h after raising the calcium of the medium to the normal level, intense binding of PNA to cells appears and neighboring cells are connected through bundles of filaments that are fluorescently labeled by FITC-insulin. After 2 days in normal calcium medium, the cultures exhibit relatively smooth, straightlined, cell boundaries that are labeled by FITC-insulin and cell boundaries and intracellular granules that are stained by hematoxylin. One day later, similar cell boundaries are present, but they are not significantly decorated by FITC-insulin and, under phase contrast microscopy, are dark. Free FITC gives labeling patterns similar to those given by FITC-insulin, but the FITC labeling is blocked by mercaptoethanol and dithiothreitol in contrast to FITC-insulin binding. The present results suggest the insulin moiety is involved in the labeling by FITC-insulin and the labeling is chronologically related to the stage of cell differentiation.

Animals↗
PubMed1986

Keratinocytes grown at the air-liquid interface.

A procedure is described which allows primary cultures of rat keratinocytes grown at the liquid-air interface to develop and maintain multilayered strata and to produce highly keratinized sheets morphologically similar to those seen in epidermis in situ. Various substrata were tested and compared as to their ability to support growth and stratification of keratinocytes. It was found that when cultured on plastic surfaces, keratinocytes adhered tightly to the substratum and produced a confluent monolayer that later stratified to two to three layers. Cells plated on Vitrogen 100 collagen failed to reach confluence and, in addition, exhibited the "clustering" phenomenon and deterioration of collagen after 3 to 4 d of growth. Significantly better attachment and spreading were observed for cells grown on rat-tail collagen as compared with plastic and Vitrogen 100 collagen. The best results, including maximal and uniform stratification, were seen in cells grown on a mixture of rat-tail and Vitrogen 100 collagens. The system that was developed in the present study offers a model for use in the study of epidermal toxicity from topically applied environmental chemicals.

Animals↗
PubMed1986

Growth and differentiation of primary rat keratinocytes on synthetic membranes.

The attachment, proliferation, and differentiation of primary cultures of keratinocytes isolated from murine epidermis were monitored after purified cell suspensions were seeded and incubated in vitro on various synthetic membranes. Concomitant studies of the effects of attachment factors added to synthetic membranes before use as substrata for keratinocytes were also done. The study demonstrated that a synthetic membrane composed of nylon was superior to other membranes and to plastic control culture vessels in supporting the growth of murine keratinocytes. Although laminin enhanced initial attachment and proliferation of cells on nylon membranes, the untreated substratum was more effective for extended incubation. Stratification and differentiation of these keratinocytes on the nylon substratum was enhanced by raising confluent cultures (7 d) to the air-medium interface so that they were in contact with medium only from the bottom. Cultures raised for 14 d produced many morphologic markers of the epidermis and closely resembled the architecture of this tissue in situ.

Animals↗
PubMed1986

Influence of adipocyte triglyceride on the partition of 2,2',4,4',5,5'-hexabromobiphenyl between 3T3L1 adipocytes and surrounding pseudoblood.

Removal from adipose tissue is an important first step in ultimate removal of many lipophilic xenobiotics from the body. This study concerned the elucidation of mechanisms by which hexabromobiphenyl (HBB) was deposited in and removed from adipocytes. Adipocytes derived from the 3T3L1 cell line of mouse fibroblasts were used to conduct studies in vitro. Results support the idea that HBB enters the 3T3L1 adipocyte via passive diffusion. A plot of the velocity of uptake versus concentration was linear, the uptake of HBB does not appear to be energy dependent, and structurally similar biphenyls did not cause an inhibition of uptake. A linear relationship between the quantities of triglyceride and HBB in the cells was found during both uptake of HBB in lipogenesis and removal of HBB in lipolysis (r greater than 0.98). This supports the contention that the quantity of triglyceride in the cells has a strong influence on the movement of HBB between adipocytes and surrounding pseudoblood. Evidence has been presented that is consistent with the hypothesis that HBB moves freely across the adipocyte membrane and is sequestered in either cells or medium according to its relative solubility in these compartments. Methods to increase the removal of HBB from adipocytes have been proposed.

Adipose Tissue↗
PubMed1985

Preliminary characterization of cell surface glycoproteins associated with epidermal differentiation in the newborn rat.

The cell surface of the newborn rat epidermis exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. It has been postulated that this change is a result of the addition of alpha-L-fucose to nonreducing terminal alpha-D-galactosyl residues on GS I-B4-binding cell surface glycoproteins. If this were the sole nature of the conversion, the physical properties of isolated GS I-B4- and UEA-bound glycoproteins should not differ greatly. UEA and GS I-B4-binding glycoproteins isolated from newborn rat epidermis have similar charge properties and are similar in the nature of their carbohydrate moieties, supporting the proposed hypothesis.

Animals↗
PubMed1985

Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies.

A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b.

Animals↗
PubMed1985

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes.

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.

Animals↗
PubMed1984

Epidermal fucosylation of cell surface glycoprotein.

When Ulex europeus agglutinin I (UEA) conjugated with fluorescein isothiocyanate is applied to tissue sections from the cutaneous epidermis of the newborn rat, the lectin binds to the surfaces of cells in the layer immediately above the basal layer but not to the cells in the basal layer itself. The latter cells bind the isolectin I-B4, from Griffonia simplicifolia (GS I-B4). The addition of a fucosyl residue to the oligosaccharide of the glycoprotein found on the surface of the basal cell can account for the change in lectin-binding specificity which occurs as the basal cell moves toward the cutaneous surface and becomes a spinous cell. The epidermis of the newborn rat has the necessary transferase to convert a glycoprotein with binding-specificity for GS I-B4 to binding specificity for UEA by adding a fucosyl residue from GDP-L-fucose.

Animals↗
PubMed1983

Molecular changes in the cell surface of differentiating epidermal keratinocytes.

The specific binding of lectins to the cell surfaces in particular epidermal strata indicates that oligosaccharide moieties of the glycoconjugates on the cell membrane are altered as the keratinocyte differentiates. The disappearance of binding by the Bandeiraea simplicifolia isolectin I-B4 (BS I-B4) and the appearance of binding by the Ulex europeus agglutinin I (UEA), which occur as the basal cell moves up into the spinous layer of the cutaneous epidermis of the newborn rat, could result from (a) redistribution of existing glycoconjugates in the cell membrane, (b) hydrolysis of galactosyl residues exposing fucosyl binding sites, or (c) addition of fucosyl residues to pre-existing oligosaccharides with specificity for the galactoside. Isolation of glycoproteins from the cell membrane using lectin-affinity column chromatography on Sepharose-4B showed a decrease in the amount of protein which bound to the BS I-B4 and an increase in the protein with affinity for the UEA as the ratio of spinous to basal cells increased in the population of cells from which the glycoproteins were obtained. These data indicate the existence of two different classes of glycoproteins on the surfaces of cells in the lower epidermis and argue against a redistribution of glycoconjugates as the explanation for the selective binding of specific lectins observed in tissue sections. Exposure of sections of skin to alpha-galactosidase completely eliminated the binding of fluorescein isothiocyanate-labeled (FITC) BS I-B4 in the lower epidermis but did not result in additional binding of UEA. Exposure to fucosidase eliminated binding by UEA and increased the binding of BS I-B4 in the spinous layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Modification of cell surface glycoprotein: addition of fucosyl residues during epidermal differentiation.

When cutaneous sections from the newborn rat were treated with alpha-fucosidase, Ulex europeus agglutinin I (UEA) binding to the cell surface of the differentiated cells in the epidermis was diminished and there was an appearance in these cell layers of binding by Bandeiraea simplicifolia I-B4 lectin (BS I-B4), which normally is specific for the basal cells. A similar treatment with alpha-galactosidase resulted in a loss of BS I-B4 binding, but had no effect on UEA binding. Glycoproteins isolated from the membranes of epidermal cells showed a threefold increase in the ratio of binding to UEA versus BS I-B4 affinity columns as the proteins were derived from the more differentiated cell populations. These data suggest that alpha-fucosyl residues are added to the glycoproteins on the cell surfaces of differentiated cells, thus blocking alpha-galactosyl residues and changing the lectin binding specificity as epidermal cells move out of the basal cell layer.

Animals↗
PubMed1981

Reversible microsomal binding of hepatic aldolase.

Fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) partitions between the microsomes and the cytosol when a rat liver homogenate is fractionated by differential centrifugation. Gel electrophoresis and immunodiffusion indicate that the one isozyme present in the liver of the young adult rat is found in both fractions. The association of the aldolase with membranes is differentially sensitive to a variety of metabolites and inorganic salts. In the absence of cellular salts, 1 mM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate elutes 50% of the enzyme from the microsomes. About 9 mM Pi or citrate is necessary to produce the same effect. With other metabolites or inorganic salts higher concentrations are required. The fraction of total enzyme which partitions with the microsomes when a homogenate is submitted to high speed centrifugation, correlates inversely with the level of fructose 1,6-bisphosphate in the supernatant solution and this concentration is higher when the tissue concentration in the homogenate is greater. The Km for fructose 1,6-bisphosphate of 3 . 10(-4) for aldolase bound to microsomes is decreased to 6 . 10(-6) M when the enzyme is dissociated from the membranes with salt. These observations appear relevant to the ongoing discussion regarding the physiological relevance of the subcellular localization of glycolytic enzymes.

Animals↗
PubMed1981

Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis.

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.

Animals↗