Isadore A. Bernstein

Keratinocytes grown at the air-liquid interface

SummaryA 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.

Growth and differentiation of primary rat kerationocytes on synthetic membranes

SummaryThe attachment, proliferation, and differentiation of primary cultures of kerationcytes 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 kerationcytes 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.

Effect of light and media upon growth and melanin formation in aureobasidium pullulans (de By.)Arn. (=pullularia pullulans)

Polysaccharides like dextrine and starch are shown to be the best carbon sources for the growth ofAureobasidium pullulans although growth is good upon a variety of other carbon sources. Light increases growth markedly when polysaccharides are the carbon source but not when other sugars are used. Variation in cell morphology is described in response to sugars and light. Extracellular granules, whose properties resemble those of melanin, are produced when dextrine is the carbon source in a defined medium containing asparagine as the source of nitrogen. The dark pigment was extracted from the walls of thick-walled brown cells ofA. pullulans and characterized as a melanin on the basis of several tests, including solubility and absorption spectrum.A. pullulans was grown on several defined and undefined media and the response of the fungus to light is shown to be determined by the medium, and the temperature at which the cultures are grown.

Nature of the repair process associated with the recovery of Escherichia coli after exposure to Cd2

When different strains of Escherichia coli are exposed to Cd2+, the cells accommodate after a long lag and proliferate. The time required for this response depends on the nature of the strain and the supplements in the growth medium. Immediately after exposure to Cd2+, considerable single strand breaks in the DNA are observed but the DNA is repaired prior to the initiation of cell proliferation. The finding that accommodation occurs in DNA polymerase I-deficient mutant cells suggests that DNA polymerase I may not be required for repair of damaged DNA in Cd2+-exposed cells. The recovery of Cd2+-exposed cells in a temperature-sensitive DNA ligase mutant cells at the permissive temperature (30° C) and failure to recover at the non-permissive temperature (42° C) indicates, however, that DNA ligase is involved in the repair of the single strand breaks associated with Cd2+-induced damage.

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.

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.

Restoration of hepatic mitochondria during recovery from carbon tetrachloride intoxication

Abstract During CCl 4 intoxication in rats, a disruption of hepatic mitochondrial structure and function occurs, which is characterized by a loss of respiratory activity, loss of phosphorylation coupled to respiration, and mitochondrial swelling, attended by loss of cristae structure. Within 15–25 hr, after full development of the mitochondrial lesion, the function and structure of the mitochondria are largely restored. Studies of the turnover of mitochondrial DNA and the rates of synthesis of mitochondrial DNA and protein indicated that the CCl 4 -insulted hepatocyte is repairing the mitochondrial damage by the insertion of specific elements into the damaged organelle, rather than by proliferation of undamaged mitochondria for replacement. The failure of ethidium bromide, oxytetracycline and chloramphenicol, specific inhibitors of mitochondrial protein, and/or nucleic acid synthesis, to block this restoration substantiates the postulated repair process, and also indicates the non-critical nature of the respective mitochondrial functions during the repair process. Cytochrome measurements made during the period of acute damage revealed normal levels of cytochrome c, c 1 and aa 3 . The observed elevation of cytochrome b is attributed to contamination of the preparation by hemoglobin.

Biosynthesis, in vitro, of “histidine-protein” — A biochemical marker in epidermal differentiation

Abstract Unusual protein containing a high level of histidine and eight other amino acids ( i.e. glycine, alanine, serine, threonine, arginine, glutamic acid, aspartic acid and tyrosine) is localized in the epidermal granular cell layer of the newborn rat. It appears that the formation of “histidine-protein” is a step in the sequence of epidermal differentiation terminating in keratinization. This paper reports the biosynthesis of “histidine-protein” by granular cells, in vitro . The protein, having a molecular weight of about 30 000, is obtained by extraction of homogenized epidermis in 8 M urea, dialysis of the extracted protein, lyophilization of the non-dialyzable material, extraction of the dried residue at 24° with 0.1 M HClO 4 , precipitation at pH 4.5 and purification on Sephadex G-100. Synthesis of “histidine-protein” appears to occur in the “large” microsomal fraction and to involve at least two steps. A “peptide precursor”, formed initially in a reaction sensitive to puromycin, is incorporated into “histidine-protein” by a mechanism not blocked by this inhibitor.

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10-5 M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of the cells with a microfilament-disrupting drug, cytochalasin B (10 μg/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5–15 min. A morphological abnormality, microtubule paracystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.

Assessment of the role of DNA damage and repair in the survival of primary cultures of rat cutaneous keratinocytes exposed to bis(2-chloroethyl)sulfide

Toxicity manifests itself as vesication in human skin exposed topically to bis(2-chloroethyl)sulfide (BCES). The destruction of the proliferating population of epidermal cells is a major component of the pathogenic process. Available data strongly suggest that damage to cellular DNA is a critical factor in the loss of these cells. However, the influence of DNA repair on this toxic response has not been adequately studied. Therefore, a study was undertaken to ascertain the influence of DNA repair on the survival of primary monolayer cultures of rat cutaneous keratinocytes exposed to BCES. The sensitive nucleoid sedimentation assay was employed for the determination of DNA damage in cultures exposed to very low levels of BCES. Initial experiments demonstrated that within 1 hr of exposure to as little as 0.1 microM BCES the structural integrity of cellular DNA was compromised, presumably resulting from the appearance of single-strand breaks in the nucleic acid. This same effect was demonstrated in basal cells derived from a stratified, cornified culture grown at the air-liquid interface and exposed topically to the vesicant. Further studies with the monolayer culture demonstrated that the gross structural integrity of the DNA in cells exposed to as much as 5 microM BCES was completely restored within the first 22 hr following the exposure. However, this repair process appeared to be inefficient since a depression of thymidine incorporation into DNA and a significant loss of DNA were exhibited in exposed cultures as long as 72 hr after the initial exposure.