White sand vegetation in an Amazonian lowland under the perspective of a young geological history.

What controls the formation of patchy substrates of white sand vegetation in the Amazonian lowlands is still unclear. This research integrated the geological history and plant inventories of a white sand vegetation patch confined to one large fan-shaped sandy substrate of northern Amazonia, which is related to a megafan environment. We examined floristic patterns to determine whether abundant species are more often generalists than the rarer one, by comparing the megafan environments and older basement rocks. We also investigated the pattern of species accumulation as a function of increasing sampling effort. All plant groups recorded a high proportion of generalist species on the megafan sediments compared to older basement rocks. The vegetation structure is controlled by topographic gradients resulting from the smooth slope of the megafan morphology and microreliefs imposed by various megafan subenvironments. Late Pleistocene-Holocene environmental disturbances caused by megafan sedimentary processes controlled the distribution of white sand vegetation over a large area of the Amazonian lowlands, and may have also been an important factor in species diversification during this period. The integration of geological and biological data may shed new light on the existence of many patches of white sand vegetation from the plains of northern Amazonia.

Hurricanes are limiting the mangrove canopy heights in the Gulf of Mexico.

Mangrove canopy height (MCH) has been described as a leading characteristic of mangrove forests, protecting coastal economic interests from hurricanes. Meanwhile, winter temperature has been considered the main factor controlling the MCH along subtropical coastlines. However, the MCH in Cedar Key, Florida (∼12 m), is significantly higher than in Port Fourchon, Louisiana (∼2.5 m), even though these two subtropical locations have similar winter temperatures. Port Fourchon has been more frequently impacted by hurricanes than Cedar Key, suggesting that hurricanes may have limited the MCH in Port Fourchon rather than simply winter temperatures. This hypothesis was evaluated using novel high-resolution remote sensing techniques that tracked the MCH changes between 2002 and 2023. Results indicate that hurricanes were the limiting factor keeping the mean MCH at Port Fourchon to <1 m (2002-2013), as the absence of hurricane impacts between 2013 and 2018 allowed the mean MCH to increase by 60 cm despite the winter freezes in Jan/2014 and Jan/2018. Hurricanes Zeta (2020) and Ida (2021) caused a decrease in the mean MCH by 20 cm, breaking branches, defoliating the canopy, and toppling trees. The mean MCH (∼1.6 m) attained before Zeta and Ida has not yet been recovered as of August 2023 (∼1.4 m), suggesting a longer-lasting impact (>4 years) of hurricanes on mangroves than winter freezes (<1 year). The high frequency of hurricanes affecting mangroves at Port Fourchon has acted as a periodic "pruning," particularly of the tallest Avicennia trees, inhibiting their natural growth rates even during quiet periods following hurricane events (e.g., 12 cm/yr, 2013-2018). By contrast, the absence of hurricanes in Cedar Key (2000-2020) has allowed the MCH to reach 12 m (44-50 cm/yr), implying that, besides the winter temperature, the frequency and intensity of hurricanes are important factors limiting the MCH on their latitudinal range limits in the Gulf of Mexico.

Assessment the Impacts of Sea-Level Changes on Mangroves of Ceará-Mirim Estuary, Northeastern Brazil, during the Holocene and Anthropocene.

Predictions of the effects of modern Relative Sea-Level (RSL) rise on mangroves should be based on decadal-millennial mangrove dynamics and the particularities of each depositional environment under past RSL changes. This work identified inland and seaward mangrove migrations along the Ceará-Mirim estuary (Rio Grande do Norte, northeastern Brazil) during the mid-late Holocene and Anthropocene based on sedimentary features, palynological, and geochemical (δ13C, δ15N, C/N) data integrated with spatial-temporal analysis based on satellite images. The data indicated three phases for the mangrove development: (1°) mangrove expansion on tidal flats with estuarine organic matter between >4420 and ~2870 cal yrs BP, under the influence of the mid-Holocene sea-level highstand; (2°) mangrove contraction with an increased contribution of C3 terrestrial plants between ~2870 and ~84 cal yrs BP due to an RSL fall, and (3°) mangrove expansion onto the highest tidal flats since ~84 cal yr BP due to a relative sea-level rise. However, significant mangrove areas were converted to fish farming before 1984 CE. Spatial-temporal analysis also indicated a mangrove expansion since 1984 CE due to mangrove recolonization of shrimp farming areas previously deforested for pisciculture. This work mainly evidenced a trend of mangrove expansion due to RSL rise preceding the effects of anthropogenic emissions of CO2 in the atmosphere and the resilience of these forests in the face of anthropogenic interventions.

Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system.

A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.

Development of an oral mucosa equivalent using a porcine dermal matrix.

We evaluated the suitability of a porcine acellular dermal matrix for the development of a 3-dimensional oral mucosal equivalent using an ex vivo-produced oral mucosal equivalent (EVPOME). Oral keratinocytes were seeded in a submerged model, and then in an air-liquid interphase model, using Transwell® inserts. EVPOME showed good cell viability and increased glucose consumption over time. Histological evaluation showed that stratified differentiated epithelium had formed in all matrices.

Biochemical indicators of implantation success of tissue-engineered oral mucosa.

Real-time (RT) determination of the health of in vitro tissue-engineered constructs prior to grafting is essential for prediction of success of the implanted tissue-engineered graft. In addition, the US Food and Drug Administration requires specific release criteria in RT prior to the release of tissue-engineered devices for human use. In principle, assessing the viability and functionality of the cellular component can be achieved by quantifying the secretion of growth factors and chemokines of tissue-engineered constructs. Ex vivo-produced oral mucosa equivalents (EVPOMEs) were fabricated under thermally stressed conditions at 43 °C for 24 h to create a functionally compromised EVPOME. We used microchannel enzyme-linked immunosorbent assay to evaluate the functionality of the cellular component, oral keratinocytes, of stressed and unstressed EVPOMEs by measuring the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) into the spent medium, which was collected on the same day prior to graft implantation into severe combined immunodeficiency mice. Implanted EVPOMEs' histology on the seventh postimplantation day was used to correlate outcomes of grafting to secreted amounts of IL-8, hBD-1, VEGF, TIMP-1, and TIMP-2 from corresponding EVPOMEs. Our findings showed that significantly higher levels of IL-8, hBD-1, and TIMP-2 were secreted from controls than from thermally stressed EVPOMEs. We also found a direct correlation between secreted VEGF and IL-8 and blood vessel counts of implanted EVPOMEs. We concluded that measuring the constitutive release of these factors can be used as noninvasive predictors of healthy tissue-engineered EVPOMEs in RT, prior to their implantation.

Epidermal keratinocyte growth: changes in protein composition and synthesis of keratins in differentiating cultures.

The epidermal skin layer undergoes extensive changes in all areas of metabolism as the lowermost basal cell differentiates into a dead structure that forms the upper, protective stratum corneum. Epidermal keratinocyte differentiation was studied in vitro using primary epidermal keratinocyte cultures that grow from a basal cell monolayer to a differentiating and proliferating, multilayered keratinocyte structure. Specific groups of proteins that form complex structures as keratinocyte differentiation occurs were studied. The quantities and synthesis of the proteins forming the tonofilamentous bundles, keratohyaline granules, and cornified cell envelopes in these cultures were determined. The selective solubilities of these proteins in a series of buffers were exploited to separate the proteins into 6 fractions. Protein assays, [3H]-amino acid pulse labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were used to quantitate and characterize the proteins extracted from basal monolayers and from stratifying and fully differentiated keratinocyte cultures. The results showed that multilayered, more differentiated cultures accumulated the greatest amount of keratin, keratohyaline granule, and cell envelope proteins, although there was no apparent increase in the synthesis of these proteins in the more differentiated cultures. These cultures showed extensive disulfide cross-linking of the keratins. Covalent keratin bonding occurred at least 6 hr after the synthesis and rapid noncovalent bonding of the polypeptides into keratins; thus various stages of keratin formation were identified. The differentiation of the epidermis appeared to be a complex, orderly, and regulated process that can be studied in vitro using this epidermal cell culture system.

Cyclic AMP, glucocorticoid, and retinoid modulation of in vitro keratinocyte growth.

The hypothesis that an imbalanced cyclic AMP and cyclic GMP ratio was central to the cutaneous expression of psoriasis prompted the design of a series of in vitro experiments. The aim of these studies was to describe the functional effects of increased intracellular cyclic AMP and of drugs therapeutic in psoriasis on epidermal keratinocyte growth. Epidermal basal cells trypsinized from neonatal mouse and adult and neonatal human skin were grown on plastic or on gelled collagen surfaces. These were used to study the effect of cyclic AMP analogues and cholera toxin (an irreversible stimulator of cyclic AMP synthesis) on keratinocyte growth. Greatly increased intracellular cyclic AMP levels, that is, 60-fold to 70-fold, stimulated neonatal mouse keratinocyte proliferation and differentiation; these same doses were cytotoxic to both neonatal and adult human cells. However, modest increases in intracellular cyclic AMP did stimulate adult human keratinocyte proliferation. The glucocorticoid triamcinolone acetonide inhibited neonatal mouse keratinocyte proliferation for approximately 1 week; the cells then became refractory to the triamcinolone acetonide effect. Triamcinolone acetonide did not apparently act through cyclic AMP-mediated events. In fact, this glucocorticoid inhibited cyclic AMP-stimulated epidermal keratinocyte proliferation. Likewise, vitamin A analogues, including the psoriasis therapy drug Ro 10-9359 inhibited neonatal mouse keratinocyte proliferation and specific differentiation events; the retinoids therapeutic in psoriasis apparently did not act via cyclic AMP-mediated events and inhibited cyclic AMP-stimulated functions. Our results indicated that cyclic AMP is a mitogenic signal for epidermal keratinocytes. This cyclic nucleotide may be important in regulating epidermal hyperproliferation. A central role for cyclic AMP in the cutaneous expression of psoriasis, however, is yet to be proven.

Retinoic acid stimulates essential fatty acid-supplemented human keratinocytes in culture.

The effect of all-trans retinoic acid on the proliferation of essential fatty acid (EFA)-deficient and of EFA-supplemented adult human keratinocytes was investigated. EFA-deficient cell strains were supplied with one of four different fatty acid-supplemented media at the P0 to P1 passage. All-trans retinoic acid at 0.5 or 1.0 microM was added to the cultures at the P1 to P2 passage. At passage P3, and 3 and 7 d thereafter, the cell growth rate was determined. The fatty acid content of cultures grown in each medium was measured using gas chromatography. All the EFA media "normalized" the cellular fatty acid composition and drastically decreased the cell number and total DNA and protein of the cultures. All-trans retinoic acid at 1 microM prevented the loss of cell viability and growth usually associated with EFA supplementation but did not affect the control (EFA deficient) or 18:1 fatty acid-supplemented cultures. All-trans retinoic acid at 1 microM altered the fatty acid content of the EFA-supplemented cultures. A statistically significant increase in 14:0, 14:1, 16:1, 18:1, and 20:4 fatty acids occurred, whereas the amounts of 18:0 and 18:2 fatty acids decreased. The largest changes were in 16:1 fatty acid (8-14%) and 18:2 fatty acid (12-5%). All-trans retinoic acid at 0.5 microM also affected both cell growth and fatty acid composition without induction of the CRABP II message. These studies demonstrate that all-trans retinoic acid stimulates the growth of EFA-supplemented keratinocyte cultures while also altering the fatty acid composition of the cells.

Increased prostaglandin synthesis by low calcium-regulated keratinocytes.

The effect of calcium concentration on the in vitro prostaglandin production by murine keratinocytes was studied using radioimmunoassay. Keratinocytes grown in low-calcium medium (0.02 mM) maintained intracellular calcium levels adequate for arachidonic acid metabolism and actually showed increased prostaglandin production. Baseline, unstimulated PGE2 production was 4.5 times higher in cells growing in low- compared to normal-calcium (1.2 mM) medium (p = 0.001). PGF2 alpha production was increased 2.5 times in the low-calcium cells (p = 0.002). The calcium ionophore A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibited differing calcium requirements for activation of the arachidonic acid pathway. A23187 ionophore stimulated prostaglandin synthesis only in cells growing in normal-calcium medium while TPA stimulated prostaglandin production by both low- and normal-calcium cells. Paradoxically, short-term exposure of low calcium-grown cells to normal-calcium medium abolished the TPA effect. These results suggested that calcium can control arachidonic acid metabolism at a number of regulatory points.

Partial characterization of phospholipase C activity in normal, psoriatic uninvolved, and lesional epidermis.

Arachidonic acid (AA), the precursor of prostaglandins and leukotrienes, can be directly liberated from membrane phospholipids by phospholipase A2 or indirectly by phospholipase C. One or both of these enzymes may be responsible for the increased content of AA found in psoriatic lesional epidermis. Keratome biopsies were obtained from normal and psoriatic individuals. After homogenization and sonication, a 10,000 g supernatant was used as the enzyme source. The activities of both phospholipase A2 and C were assayed in each sample using phosphatidylcholine and phosphatidylinositol, respectively, as substrates. Phospholipase A2 activity was found to be significantly higher than normal in both uninvolved and lesional psoriatic epidermis. In contrast, phospholipase C activity was significantly higher than normal in only the psoriatic plaque on the basis of wet weight (p less than 0.001), protein (p = 0.01), and DNA (p = 0.004) content. Phospholipase C activity in pmol diacylglycerol formed/min/microgram DNA was: normal 4.96 +/- 0.80, n = 13; uninvolved 7.29 +/- 1.06, n = 18; plaque 14.44 +/- 2.50, n = 18. Analysis (pH profile, calcium requirement, substrate specificity, and saturation kinetics) of pooled epidermal extracts showed no inherent differences in phospholipase C from normal and psoriatic epidermis, suggesting either a higher concentration or the presence of an activated form of the enzyme in psoriatic plaque. Since phospholipase C activity, in contrast to phospholipase A2 activity, is elevated only in lesional epidermis, it is possible that this enzyme contributes to AA accumulation observed in this tissue.

Formation of epidermal cell thymocyte-activating factor (ETAF) from cultured human keratinocytes from normal and uninvolved psoriatic skin.

Cultures of keratinocytes from uninvolved psoriatic skin have been shown to exhibit increased DNA synthesis. Since epidermal cell-derived thymocyte-activating factor (ETAF) can stimulate the proliferation of human keratinocytes in vitro, we have compared the formation of ETAF in cultured keratinocytes from normal and uninvolved psoriatic skin. Trypsinized epidermal cells were plated at nonconfluent concentrations in dishes coated with a collagen type I gel. Normal keratinocyte cultures spontaneously released ETAF into the supernatant from day 1, reaching a maximum level (mean 124 +/- 4 units/micrograms DNA) on day 7, just before the cultures became completely confluent (days 9-12). The ETAF release then gradually decreased until a plateau (mean 16 +/- 7 units/micrograms DNA) was reached on day 18. In normal keratinocyte cultures the incorporation of [3H]thymidine into DNA changed in parallel with the ETAF release. Psoriatic keratinocytes had normal rates of ETAF release and of DNA synthesis until the time of culture confluency. However, when complete confluency was obtained, psoriatic keratinocytes continued to synthesize DNA at high levels (increased by 146% on day 15) while their ETAF release declined. Analysis of intracellular ETAF showed a significant correlation with the extracellular ETAF in cultures from both normal (r = 0.77, n = 10, p less than 0.02) and uninvolved psoriatic skin (r = 0.76, n = 12, p less than 0.01). These results indicate a relation between ETAF formation and DNA synthesis of normal keratinocyte cultures. However, factors other than ETAF appear to be responsible for the elevated DNA synthesis of confluent keratinocyte cultures from uninvolved psoriatic skin.

Normalization of essential-fatty-acid-deficient keratinocytes requires palmitic acid.

Cultured adult human keratinocytes show accelerated growth rates in medium that is essential fatty acid deficient. The cells also show decreased amounts of the essential fatty acids 18:2, 20:3, and 20:4 and contain increased amounts of the monounsaturated fatty acids 16:1 and 18:1. These lower levels of polyunsaturated fatty acids were only partially restored by supplementing the medium with 18:2 and 20:4 fatty acid. The addition of the non-essential fatty acid 16:0 (5 microM), along with the essential fatty acids, resulted in the successful normalization of the major fatty acids in the deficient keratinocytes. Normalized cells showed a constant total fatty acid/mg of protein in the phospholipid fraction, as the total cell fatty acid content per cell increased with augmenting fatty acid supplementation. Supplementation of the medium with 16:0 and essential fatty acids decreased the growth and passage potential of the cells. Use of 18:1 in lieu of 18:2 fatty acid yielded essential-fatty-acid-deficient keratinocyte growth values. Likewise the least supplemented medium (5 microM 18:2 + 5 microM 16:0) also gave the accelerated cell growth rates. This study shows that manipulation of the essential fatty acid levels, if accompanied by 5 microM 16:0 in the growth medium, alters the growth properties of adult human primary keratinocytes.

Cyclic AMP, cyclic GMP, and glucocorticoids as potential metabolic regulators of epidermal proliferation and differentiation.

The two cyclic nucleotides, cyclic AMP and cyclic GMP, appear to be central to the metabolic regulation of cell proliferation and differentiation in various cells. Moreover, in many systems glucocorticoids appear to act in concert with or parallel to cyclic AMP. The available evidence suggests that these three molecular species--cyclic AMP, cyclic GMP, and glucocorticoids--may be essential to the normal regulation of epidermal proliferation and differentiation. In 1970, we suggested that perturbed epidermal homeostasis, exemplified by psoriasis, might be associated with low cellular levels of cyclic AMP and, in 1972, with high levels of cyclic GMP as well. Subsequent measurements of these two cyclic nucleotides in our laboratory showed a probable reduction in the cyclic AMP/cyclic GMP ratio in lesional psoriatic tissue. This led to the hypothesis that the cardinal features of psoriatic epidermis--glycogen accumulation, excessive proliferation, and reduced cell specialization--are the results of this reduced ratio. A corollary of this hypothesis was that a psoriatic lesion could not begin or exist without this altered cyclic nucleotide ratio. Recently, four different agents--lithium, a beta adrenergic blocking agent, antimalarials, and iodide--have been found to exacerbate psoriasis and to reduce the formation of cyclic AMP in various tissues. Consequently we believe that cyclic nucleotides are of central importance in the pathogenesis of the epidermal component of psoriasis.

Clotrimazole, an inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation, and macromolecular binding in BALB/c mouse cultured keratinocytes.

The effect of the antifungal imidazole compound, clotrimazole, on the metabolism of benzo[a]pyrene (BP) was studied in cultured keratinocytes prepared from BALB/c mouse epidermis. Varying concentrations of clotrimazole added to the cultured keratinocytes resulted in a dose-dependent inhibition of the activities of the microsomal cytochrome P-450-dependent monooxygenases aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The major organic solvent-soluble metabolites of BP identified in the cultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol), 9-hydroxybenzo[a]pyrene (9-OH-BP), and 3-hydroxybenzo[a]pyrene (3-OH-BP), although small amounts of trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, BP-quinones, and trans-9,10-dihydroxybenzo[a]pyrene were also present. The major organic solvent-extractable metabolites of BP found in the extracellular culture medium were primarily the diols with smaller quantities of phenols and quinones. The major water-soluble metabolites of BP present both intracellularly and extracellularly were glucuronide conjugates of 3-OH-BP, 9-OH-BP, and benzo[a]pyrene-3,6-dione and to a lesser extent sulfate conjugates (primarily of the BP-7,8-diol). Clotrimazole inhibited the generation of organic solvent-soluble and water-soluble conjugates in a dose-dependent manner. The in vitro metabolism of BP by microsomes prepared from control and benz[a]anthracene (BA)-induced cultured keratinocytes was also inhibited by clotrimazole with greater inhibitory effect on BA-induced keratinocytes especially with respect to the formation of diols and quinones. The enzyme-mediated covalent binding of BP to mouse keratinocyte DNA and protein was also substantially diminished by clotrimazole in a dose-dependent fashion. These results indicate that clotrimazole, a widely used drug for the management of a variety of superficial dermatophyte infections of the skin, is a potent inhibitor of cytochrome P-450-dependent transformation of polycyclic aromatic hydrocarbons in cultured murine keratinocytes. This system offers a convenient approach for studies as inhibitors of carcinogen metabolism in the epidermis.

Decreased protein kinase C activity in psoriatic versus normal epidermis.

To study the possible involvement of protein kinase C in psoriasis, we determined the activity of this enzyme in involved and uninvolved epidermis from psoriasis patients as well as in normal epidermis from controls. Protein kinase C activity was measured in the 100,000 g supernatants and in the detergent-solubilized particulate fractions of tissue homogenates after partial purification by DEAE-cellulose chromatography. The total enzyme activities per gram protein for normal, involved, and uninvolved epidermis were 127 +/- 11.9 mU/g, 64.7 +/- 8.6 mU/g, and 88.5 +/- 14.4 mU/g, respectively. The respective values for total enzyme activity per mg DNA were 7.04 +/- 0.48 mU/mg, 4.28 +/- 0.54 mU/mg, and 5.02 +/- 0.66 mU/mg. By both data bases, protein kinase C activity was statistically lower in the psoriatic skin biopsies than in those from control persons, whereas no significant difference was found between involved and uninvolved epidermis from psoriasis patients. We hypothesize that alterations in protein kinase C-mediated processes due to decreased protein kinase C activity may play a significant role in the pathophysiology of psoriasis.

In vitro model of essential fatty acid deficiency.

The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA)-deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty acids 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85-90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid-deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure.

Biosynthesis of lipoxygenase products by enzyme preparations from normal and psoriatic skin.

Incubations of [14C]arachidonic acid [( 14C]AA) with cell-free preparations from normal, clinically involved and uninvolved epidermis from psoriatic subjects resulted in the formation of several radiolabeled metabolites of the lipoxygenase pathway. The identities of the monohydroxy-ETEs and dihydroxy-ETEs (products of the 12-lipoxygenase and 5-lipoxygenase pathways) were determined by comparison with authentic standards of 12L-hydroxy-5,8,10,14-eicotetraenoic acid (12-HETE) and authentic 5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB4) by thin-layer chromatography in two solvent systems; by silicic acid column chromatography and by normal phase and straight phase high-pressure liquid chromatography. Activity of the enzymes which catalyze this transformation are localized in the soluble (105,000 g supernatant) fraction of the epidermal preparations. The activity of enzymes of both pathways were inhibited by 5,8,11,13-eicosatetraynoic acid (ETYA) and nor-dihydroguaretic acid (NDGA), known inhibitors of the lipoxygenase and cyclooxygenase pathways. Transformation of [14C]AA into [14C]LTB4-like metabolite by the soluble preparations from clinically involved psoriatic epidermis was significantly higher (p less than 0.001) than from paired uninvolved soluble preparations or from soluble preparations from normal subjects. Furthermore, biosynthesis of LTB4-like metabolite by the uninvolved soluble preparation was significantly higher (p less than 0.05) than preparations from normal epidermis. These results imply that the [14C]LTB4-like metabolite biosynthesized by the clinically involved soluble preparation was due at least in part to the increased activity of the lesional enzymes and not entirely due to possible intraepidermal infiltrating neutrophils. Human epidermal preparations, therefore, contain enzymes which catalzye the transformation of labeled AA into labeled LTB4-like metabolite as well as into other yet unidentified dihydroxy-ETEs. Localization of a soluble 5-lipoxygenase-like activity in the epidermis implies a possible role of the lipoxygenase products in the proliferative and inflammatory processes in this tissue.

Aryl hydrocarbon hydroxylase, epoxide hydrolase, and benzo[a]-pyrene metabolism in human epidermis: comparative studies in normal subjects and patients with psoriasis.

Prior studies have shown that human skin possesses a cytochrome P-450-dependent microsomal enzyme that is capable of metabolizing drugs and polycyclic aromatic hydrocarbon (PAH) carcinogens. This study characterized benzo[a]pyrene (BP) metabolism in human epidermis of normal and psoriatic individuals. The basal level of the cytochrome P-450-dependent microsomal enzyme aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH) were measured in freshly keratomed epidermis from 12 normal individuals and from uninvolved skin sites of 12 patients with psoriasis. The induction response of AHH following the in vitro addition of the PAH benz[a]anthracene (BA) was also assessed. The basal activity (mean +/- SE) of AHH in normal epidermis was 62.1 +/- 5.6 units (fmol 3-hydroxybenzo[a]pyrene, 3-OH-BP/min/mg protein) whereas the activity in uninvolved skin of psoriatic individuals was 62.9 +/- 5.1 units (NS), Epoxide hydrolase activity was 25.1 +/- 1.1 (pmol BP 4,5-diol/min/mg protein) unites in normal epidermis and 24.8 +/- 2.1 units in epidermis from patients with psoriasis (NS). Following addition of BA (100 microM), in vitro, AHH activity in normal epidermis increased by a mean value of 165% whereas activity in nonlesional epidermis of psoriatic individuals increased 320%. Kinetic studies in normal epidermis revealed that AHH reaction was linear up to 60 min and to 50 micrograms protein, had a pH optimum of 7.4, and the Km for BP was 0.62 microM. High-performance liquid chromatography (HPLC) confirmed that the pattern of metabolism of BP was quite similar in epidermal microsomes prepared from normal and psoriatic individuals, insofar as the formation of diols, phenols, and quinones was concerned. These studies indicate that human epidermis is capable of metabolizing BP and that there is no significant difference between normal individuals and patients with psoriasis insofar as basal AHH activity or total BP metabolism is concerned. Furthermore, the epidermal enzyme system in patients with psoriasis has a greater responsiveness to environmental PAH than does that of normal individuals.

Oleic acid and linoleic acid are the major determinants of changes in keratinocyte plasma membrane viscosity.

Keratinocytes were grown in medium with no essential fatty acids as well as in media with specially selected fatty acid augmentations. Gas chromatographic determinations of 21 fatty acids in the phospholipids were correlated with plasma membrane viscosity obtained by electron paramagnetic resonance studies (n = 24). Using standard procedures from multivariate analysis, we derived an expression that modeled the viscosity data as a function of four key fatty acid levels: [formula see text] where the fatty acids are given in mole percent of total lipids and are identified as two number sequences: number of carbons followed by number of double bonds. No other fatty acid made a significant contribution to the regression equation. The range of viscosity was very large, varying from 60 to 120 cP over the sample population. The results are interpreted to indicate that polyunsaturated fatty acids are replaced with monounsaturated fatty acids by the keratinocytes and that dihomogamma-linolenic acid (20:3, n-6) plays an important role in membrane viscosity when essential fatty acids are available in the growth medium of these adult human cultured keratinocytes.