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R R Isseroff

Publications and source records attributed to R R Isseroff.

At least 37 records · Page 2Linked to original sources
PubMed1993

Ion channels are linked to differentiation in keratinocytes.

In vivo and in vitro, keratinocyte differentiation is linked with increased extracellular Ca2+. In order to correlate ion channels with cell differentiation and investigate keratinocyte membrane responses to Ca2+, keratinocyte single channel currents were studied using the patch-clamp technique. The most frequently observed channel was a 14 pS nonspecific cation channel. This channel was permeable to Ca2+ and activated by physiological concentrations of Ca2+. We also found a 35 pS Cl- channel whose open probability increased with depolarization. Finally, a 70 pS K+ channel was seen only in cell-attached or nystatin-permeabilized patches. We correlated channel types with staining for involucrin, an early marker of keratinocyte differentiation. While the nonspecific cation channel and Cl- channel were seen in both involucrin positive and involucrin negative cells, all channels in which the K+ channel activity was present were involucrin positive. Membrane currents through these channels may be one pathway by which signals for keratinocyte proliferation or differentiation are sent.

Calcium↗
PubMed1993

Properties of enzymes hydrating epoxides in human epidermis and liver.

1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different. 2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity chromatographic properties. 3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities. 4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes. 5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts. 6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

Cytosol↗
PubMed1993

Focal dermal hypoplasia: four cases with widely varying presentations.

We describe four patients with focal dermal hypoplasia (FDH): a girl with classic FDH, a boy with cutaneous findings, an infant with severe multisystem disease, and the infant's mother, who had previously undiagnosed FDH. These patients illustrate the classic cutaneous manifestations of FDH and the variations that can exist within a family.

Adipose Tissue↗
PubMed1992

Quantitative in vitro assessment of phototoxicity by a fibroblast-neutral red assay.

We have adapted the neutral red uptake assay for quantitative assessment of injury to fibroblast cultures by potential phototoxins. Tetracycline derivatives, quinolone derivatives, and chlorpromazine were used as model compounds for development of the assay. Human fibroblasts were incubated with potential phototoxins, the cell cultures irradiated with UV, and the capacity for neutral red uptake determined. Demeclocycline and doxycycline, two known photosensitizers, showed a 94% and 95% decrease of neutral red uptake, respectively, indicating photo-induced cytotoxicity. Minocycline, a non-photosensitizing tetracycline derivative, showed no decrease in uptake. Tetracycline, a weak phototoxin, showed minor (10%) decrease at equivalent concentrations (20 micrograms/ml). Microscopic observation of neutral red uptake and cell damage paralleled the spectrophotometric findings. Chlorpromazine, a non-tetracycline phototoxin, showed 91% decrease. An additional group of phototoxic drugs, quinolone antibacterials, were studied. Nalidixic acid, ofloxacin, ciprofloxacin, and norfloxacin all demonstrated phototoxicity, with nalidixic acid showing the greatest decrease in neutral red uptake. This methodology may provide a useful rapid method to quantify phototoxic potential of new drugs or suspected phototoxins.

Cells, Cultured↗
PubMed1992

Low-energy helium neon laser irradiation does not alter human keratinocyte differentiation.

There are reports that low-energy HeNe irradiation can enhance wound healing in vivo. We have previously demonstrated that HeNe irradiation increases the motility of human epidermally derived keratinocytes in vitro. Here we investigate whether HeNe irradiation alters normal keratinocyte differentiation, which is essential for the formation of a normal, functioning epidermis. Subconfluent keratinocyte cultures were irradiated three times within 24 h with either 0, 0.8, 3, or 7.2 J/cm2. After cultures reached post-confluence, parameters of growth and differentiation, such as cell number, cornified envelope (CE) formation, and transglutaminase activity were measured. No significant differences were found between the control (0 J) and irradiated cultures in these assays. We also examined the pattern of newly synthesized keratins in cultures irradiated with 7.2 J/cm2 three times within a 24-h period. Both control and irradiated cultures exhibited similar keratin patterns. These results provide evidence that HeNe irradiations of up to 7.2 J/cm2 have no direct deleterious effect on normal keratinocyte differentiation needed for the formation of a functional epidermis. Hence, it is anticipated that the clinical use of the HeNe laser irradiance that enhances keratinocyte migration in vitro (0.8 J/cm2) to promote wound healing in vivo will not alter the ultimate integrity or differentiated function of the epidermis that migrates to cover the wounded area.

Cell Differentiation↗
PubMed1992

Dyskeratosis congenita associated with elevated fetal hemoglobin, X-linked ocular albinism, and juvenile-onset diabetes mellitus.

An 11-year-old boy had dyskeratosis congenita, elevated fetal hemoglobin level, X-linked ocular albinism, and juvenile-onset diabetes mellitus. A review of the international literature revealed that elevated fetal hemoglobin has been noted in 15 reported cases of dyskeratosis congenita. It is a previously unrecognized, commonly associated finding in dyskeratosis congenita that may provide insight into the location and function of the gene for dyskeratosis congenita.

Albinism, Ocular↗
PubMed1990

Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.

Calcium↗
PubMed1990

Low-energy helium-neon laser irradiation increases the motility of cultured human keratinocytes.

Helium-neon (HeNe) laser irradiation is known to stimulate wound healing. We investigated whether the biostimulatory effects of HeNe irradiation result from enhancement of keratinocyte proliferation or motility. HeNe effects on keratinocyte motility were evaluated by irradiating a "wounded" culture with 0.8 J/cm2 3 times over a 20-h period. At 20 h post-irradiation, videocinemicroscopy and sequential quantitative measurements of the leading edge were taken over a 6-h period. There was a significant difference in migration of the leading edge in irradiated "wounds" compared to non-irradiated "wounded" controls (12.0 microns/h vs 4.0 microns/h, p less than 0.0001). To determine if the increase in migration observed in irradiated cultures resulted from a proliferative effect of HeNe irradiation, subconfluent human keratinocyte cultures were irradiated with single or multiple doses of different fluences of HeNe irradiation (0.4 to 7.2 J/cm2) and evaluated 72 h post-irradiation. Irradiated and non-irradiated keratinocyte cultures grown on a microporous membrane surface were co-cultured with irradiated and non-irradiated fibroblasts to determine if HeNe irradiation induced a paracrine effect on keratinocyte proliferation. No significant increase in keratinocyte proliferation was demonstrated in any of these treatments. The biostimulatory effects of HeNe irradiation may now be extended to include enhancement of keratinocyte motility in vitro; this may contribute to the efficacy of HeNe irradiation in wound healing.

Cell Division↗
PubMed1989

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes.

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.

Animals↗
PubMed1989

Plasminogen activator activity is associated with neural crest cell motility in tissue culture.

We have examined the possibility that proteases such as plasminogen activator (PA) contribute to the extraordinary motile capability of neural crest cells. We show that trunk neural crest cells that migrate from isolated neural tubes in vitro produce PA and that the level of cell-associated PA increases dramatically after 8 days in culture. This increase is not the result of differentiation or time in culture, because neural crest cell clusters that form on top of the neural tube and differentiate into pigment cells but are immotile produce very low levels of PA. If these clusters are removed from the neural tube and replated on a plastic substratum where they migrate, the level of PA associated with the cells increases dramatically, suggesting that PA production is associated with motility. Inhibitors of PA/plasmin activity significantly reduce neural crest cell motility in vitro, further supporting the idea that proteases are important in neural crest cell migration.

Animals↗
PubMed1989

Triiodothyronine alters the cornification of cultured human keratinocytes.

Scaly skin occurs in 80-90% of patients who are hypothyroid, the pathogenesis of which is unknown. Since thyroid hormone (T3) affects growth and differentiation in other organs, we examined the effects of its absence on keratinocytes in vitro. Human neonatal foreskin keratinocytes were cultivated and second passage cells were switched to T3-depleted (-T3) medium at 50% confluence. Cells maintained in the -T3 medium demonstrated increased (1.5 fold) levels of the cross-linking enzyme transglutaminase and increased (1.5 fold) formation of cornified envelopes, when compared to keratinocytes maintained in medium containing physiologic levels (2 X 10(-9)M) of T3. Additionally, in the -T3 cultures, the level of the protease plasminogen activator (PA), an enzyme implicated in the process of shedding of cornified cells, was decreased 70-80% of that measured in +T3 media. Absence of T3 from keratinocyte culture-medium increased both the level of the enzyme responsible for cross-linking cornified envelope precursors and the rate of envelope formation in cultured cells. The decreased levels of PA observed in the -T3 cultures could result in decreased shedding of cornified cells. These alterations in the process of keratinocyte differentiation may explain the clinically observed scaliness associated with hypothyroidism in humans. The molecular mechanism by which T3 alters keratinocyte cornification is not yet known.

Cell Differentiation↗
PubMed1988

Fish again for dinner! The role of fish and other dietary oils in the therapy of skin disease.

Adequate levels of polyunsaturated fatty acids are necessary for the normal functioning of most mammalian cells, both to provide fluidity to the cell membrane lipid bilayer and to function as precursors for the synthesis of the regulatory eicosanoids, prostaglandins, and leukotrienes. The omega-6 class of polyunsaturated fatty acids, such as linoleic and arachidonic acids, are of special importance as precursors for eicosanoid synthesis. The skin is a particularly good organ in which to study the effects of polyunsaturated fatty acids metabolism, inasmuch as either deficiencies of specific PUFA or overproduction of polyunsaturated fatty acids-derived prostaglandin and leukotriene result in specific, clinically recognizable cutaneous diseases. To help understand the pathogenesis of these diseases, polyunsaturated fatty acids metabolism is reviewed here, with emphasis on clinical manifestations of both deficiency syndromes and overproduction of proinflammatory eicosanoids. A rationale is presented for a therapeutic approach to inflammatory disease by dietary manipulation and substitution of omega-6 fatty acids by the unique omega- 3 class of polyunsaturated fatty acids found in fish. Evidence for the efficacy of fish oil in the therapy of specific inflammatory diseases is reviewed, as are the caveats regarding its therapeutic use. Dietary manipulations, specifically fish oil additives, appear to hold promise as therapeutic tools for cutaneous diseases.

Coronary Disease↗
PubMed1987

Novel regulatory actions of 1 alpha,25-dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes.

The in vitro incubation of murine keratinocytes in the presence of 1 alpha,25-dihydroxyvitamin D3 enhanced the rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdIns) when compared to untreated cells. The rapid hydrolysis of the polyPtdIns and the release of the inositol phosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that 1 alpha,25-dihydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular polyPtdIns; this in turn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.

Animals↗
PubMed1987

Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes.

The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo synthesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate (14C-LA) into 18:3(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) (AA) was noted. The conversion of non-radiolabeled 18:3(n-6) or 20:2(n-6) was also examined and the pattern of metabolites synthesized suggests that the preferred metabolic pathway for conversion of linoleic acid into arachidonic acid is via the classically described pathway in which a delta 6 desaturase constitutes the initial reaction. Although cultured skin fibroblasts are known to convert linoleic acid into arachidonic acid, the current study demonstrates that cultured epidermal keratinocytes can also avidly metabolize exogenous linoleic acid. The ability of cultured keratinocytes, and not of whole epidermis in vivo, to convert linoleic acid into arachidonic acid suggests that specific enzymatic activities may be induced by the tissue culture system itself. Hence, findings of metabolic capabilities in cultured cells may not necessarily be extrapolated to the in vivo situation.

Animals↗
PubMed1986

Abnormal lipogenesis in thyroid hormone-deficient epidermis.

Striking skin changes can accompany hypothyroidism, among them a scaly ichthyosis. The pathogenesis of these disorders is unknown. Since altered sterol metabolism has been associated with other scaling dermatoses, we tested the hypothesis that altered epidermal lipogenesis may accompany hypothyroidism and induce the observed scaling. Thyroidectomized (TX) rats were sacrificed 53 days postthyroidectomy, and the rate of incorporation of the radiolabeled precursor, [14C]acetate, into epidermal lipids was determined. Approximately 2-fold decreases in incorporation of precursor into both polar and neutral lipids were observed in the TX epidermis; the most significant decreases, however, were in the neutral lipids, particularly the sterol and sterol-ester moieties. Total cholesterol pool size was also reduced in the TX epidermis. Similar decreases in sterol biosynthesis were noted in human keratinocytes cultivated in medium devoid of thyroid hormone. These findings demonstrate that lipid metabolism, and notably sterol synthesis, is altered in epidermal keratinocytes deprived of thyroid hormone, and suggest that, as in other scaling disorders, this abnormal sterologenesis may lead to the clinically observed ichthyosis that can accompany hypothyroidism.

Animals↗
PubMed1985

Alterations in fatty acid composition of murine keratinocytes with in vitro cultivation.

The availability of methods for the in vitro cultivation of keratinocytes has spawned numerous studies utilizing these systems to analyze epidermal biochemical pathways, e.g., eicosanoid production. To determine whether these culture systems are indeed valid models for studies of eicosanoid products, we analyzed the fatty acid (FA) composition, especially of eicosanoid precursors linoleic acid (LA) and arachidonic acid (AA), of cultured and noncultured mouse keratinocytes. Neonatal mouse epidermal keratinocytes were cultivated in Dulbecco's modification of Eagle's medium + 10% fetal calf serum (FCS). Lipids of the cultivated cells, as well as noncultivated keratinocytes and whole epidermis were extracted in CHCl3:MeOH (2:1) and lipid classes separated by thin-layer chromatography. The FA composition of the total lipid extract as well as of the phospholipid class was determined by gas-liquid chromatography of FA methyl esters. There was a gradual decrease in the LA levels in the cultured cells; by day 5 of culture the cells demonstrated a 4-fold (p less than 0.001) decrease in LA as compared to either noncultured cells or whole epidermis. Levels of AA, on the other hand, remained unchanged during culture. Analysis of the FCS used in the culture medium revealed that the level of LA was 4-fold lower than that of normal mouse serum. Since LA is an essential FA which is not synthesized by the cell, the decreased LA in cultured cells probably results from the paucity of this FA in the FCS-containing culture medium. These studies indicate that keratinocytes cultivated in FCS-containing medium demonstrate profound alterations in levels of LA. Hence, in vitro keratinocyte studies dependent on cellular polyunsaturated FA substrates should be interpreted with caution. The relationship of altered cellular levels of LA on keratinocyte differentiation remains to be determined.

Animals↗
PubMed1985

Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.

Several problems have frustrated the isolation of lamellar bodies (LB) from mammalian epidermis. We obtained pellets enriched in intact LB by utilizing the staphylococcal epidermolytic toxin to provide intact, outer epidermal sheets, by controlled homogenization in a cell disrupter, and by passage of homogenates through a graded series of nuclepore filters (Science 221:962, 1983). Such preparations contained more intact LB than did fractions prepared by a variety of differential or sucrose/metrizamide discontinuous centrifugation methods. Initial characterization of the enzymatic content of this fraction revealed it to be enriched in certain hydrolytic enzymes (acid phosphatase, carboxypeptidase, cathepsin B, acid lipase, sphingomyelinase, and phospholipase A), but strikingly depleted in all sulfatases, beta-glucuronidase, and the non-lysosomal protease, plasminogen activator. Thus, LB show some properties of lysosomes, although certain characteristic lysosomal enzymes are strikingly absent. Lamellar body fractions contained 2-3 times more lipid per unit weight than did homogenates, and were enriched in phospholipids, free sterols, and glycosphingolipids, but not in other neutral lipids or ceramides. In summary, whereas some of the enzymes in LB could participate in the metabolism of LB lipid precursors to hydrophobic barrier constituents, others may attack intercellular constituents, ultimately resulting in desquamation. The lipid profile of these organelles suggests that they deliver precursors of permeability barrier lipids to intercellular domains.

Acid Phosphatase↗