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.

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.

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.

A putative in vitro organotypic model of molting with human skin explants.

We report finding a simple method to partially reproduce the characteristic process of molting that takes place in invertebrates using human skin explants in vitro. In this method, human skin explants discarded from regular plastic surgery procedures were kept, submersed, in regular growth medium for 10 days at 4°C. After that period, the skin explants were cultured at the air-liquid interface for another 10 days. Histological analysis of the skin revealed the formation of one full epidermal structure and an additional intermediate epidermal structure containing a putative stratum corneum, superimposed one of top of the other, in which we consider an equivalent model of "molting" or "ecdysis". Basic analysis of cell proliferation and differentiation of the explants at different stages of the process are briefly presented. We believe this model can be used in the study of certain human skin diseases as well as in comparative animal physiology.

Avaliação de duas técnicas de translocação de serrapilheirasobre as assembleias de aranhas (Arachnida: Araneae) e formigas (Hymenoptera: Formicidae)

Com o ritmo atual de destruição dos ecossistemas surge a necessidade de se investir em técnicas que visem à conservação dos remanescentes florestais e das suas espécies.Nosso objetivo foi comparar duas técnicas para translocação da serrapilheira (com e sem a retirada da camada de serrapilheira preexistente), observando como varia a riqueza e composição de aranhas e formigas no local da translocação, a fim de definir a melhor forma de realizá-la. O estudo foi realizado em dois remanescentes de Mata Atlântica (fragmento doador e receptor) localizados em Salvador (BA). Antes da translocação foi realizada uma comparação de ambas as faunas, entre os fragmentos, e foi encontradadiferença significativa tanto na riqueza quanto na composição. Assim pôde-se realizar o experimento. Para tanto, foi realizada a caracterização ambiental a fim de translocar a serrapilheira para as unidades mais similares na tentativa de minimizar o estresse dos organismos. A riqueza em espécies tanto de aranhas quanto de formigas aumentou nasunidades que receberam a serrapilheira, sendo para aranhas mais elevada nas unidades onde não foi retirada a serrapilheira preexistente. Já para as formigas as duas formas parecem eficientes. Contudo, levando em consideração os dois grupos, propõe-se que atranslocação deve ser feita mantendo a serrapilheira preexistente.

Due to the current rhythm of ecosystem destruction, the development of new techniques aiming at the conservation of forest remnants and their biota is urgent. Our objective was to compare two techniques of leaf-litter translocation (with and without withdrawing the pre-extent litter layer), through observations on spider and ant richness and assemblage composition, in order to define the best way to perform translocation. The study was carried out in two Brazilian Atlantic rain forest remnants (called releaser and receptor fragments) in Salvador, State of Bahia, Brazil. A comparison between the faunas of both fragments was performed before translocation and a significant difference was found both in their richness and composition, which is an essential condition to carry out the experiment. A small scale environmental characterization] was made in order to choose similar units capable of minimizing the stress on leaf-litter organisms living there. Both for spidersand ants, the species richness increased in units that received leaf-litter and it was higher for spiders in units where the pre-extent litter had not been removed. For the ants, the two forms of translocation seem to be efficient. Nevertheless, taking into account the two groups of organisms, it is suggested that the translocation must be done maintaining the pre-extent leaf-litter.

Model of wetland development of the Amapá coast during the late Holocene

The modern vegetation types, sedimentary sequences, pollen records and radiocarbon dating obtained from three sediment cores from Calçoene Coastal Plain were used to provide a palaeoecological history during the late Holocene of Amapá coastal wetland according to flood regime, sea-level and climatic changes. Based on these records, four phases of vegetation development are presented and they probably reflect the interaction between the flow energy to the sediment accumulation and the brackish/freshwater influence in the vegetation. This work suggests interchanges among time periods characterized by marine and fluvial influence. The longitudinal profile did not reveal the occurrence of mangrove in the sediment deposited around 2100 yr B.P. During the second phase, the mud progressively filled the depressions and tidal channels. The mangrove probably started its development on the channel edge, and the herbaceous field on the elevated sectors. The third phase is characterized by the interruption of mangrove development and the increase of "várzea" vegetation that may be due to the decrease in porewater salinity related to a decrease in marine water influence. The last phase is represented by the mangrove and "várzea" increase. The correlation between current patterns of geobotanical unit distribution and palaeovegetation indicates that mangrove and "várzea" forests are migrating over the herbaceous field on the topographically highest part of the studied coast, which can be related to a relative sea-level rise.

Os tipos de vegetação atual, sequências sedimentares, dados de pólen e datações por radiocarbono obtidas em três testemunhos de sedimento da planície costeira de Calçoene foram utilizados para estabelecer uma história paleoecológica durante o Holoceno superior das zonas úmidas costeiras do Amapá conforme as mudanças no regime de inundação, nível do mar e clima. Baseado nestes três registros, quatro fases de desenvolvimento da vegetação são apresentadas e provavelmente refletem a interação entre o fluxo de energia na acumulação do sedimento e a influência das águas salobras e doces na vegetação. Este trabalho sugere alternâncias entre períodos caracterizados por influências marinha e fluvial. O perfil longitudinal não revelou a ocorrência de manguezais nos sedimentos depositados por volta de 2100 anos A.P. Durante a segunda fase, a lama preencheu progressivamente as depressões e canais de maré. Provavelmente, os manguezais iniciaram seu desenvolvimento nas margens dos canais, e os campos herbáceos nos setores elevados. A terceira fase é caracterizada por uma interrupção no desenvolvimento dos manguezais e a expansão da vegetação de várzea devido a uma diminuição na influência das águas marinhas. A última fase é representada pela expansão de manguezais e várzeas. A correlação entre os padrões atuais de distribuição das unidades geobotânicas e a paleovegetação indica que os manguezais e as florestas de várzea estão migrando sobre os campos herbáceos nos setores topograficamente mais elevados do litoral em estudo, o que pode estar relacionado a um aumento do nível relativo do mar.

Pharmacological retention of oral mucosa progenitor/stem cells.

Oral mucosa progenitor/stem cells reside as a small-sized cell population that eventually differentiates concurrently with an increase in cell size. Activation of the mammalian target of rapamycin (mTOR) leads to an increase in cell size. We hypothesized that rapamycin, a specific inhibitor of mTOR, will maintain primary human oral keratinocytes as a small-sized, undifferentiated cell population capable of retaining their proliferative capacity. Primary, rapamycin-treated (2 nM, 20 nM) oral keratinocytes showed a diminished cell size that correlated with a higher clonogenicity, a longer-term proliferative potential, and a slower cycling cell population concurrent with decreased expression of a differentiation marker when compared with untreated cells. Only the 2-nM rapamycin-treated oral keratinocytes maintained their ability to regenerate oral mucosa in vitro after 15 weeks of culture. Rapamycin, a Food and Drug Administration-approved drug, may have applicability for use in creating a highly proliferative cell population for use in regenerative medicine.

Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture.

BACKGROUND: Cutaneous wound healing is relatively slow in patients with diabetes. OBJECTIVES: To test the hypothesis that this defect in healing of wounds in patients with diabetes results from dysfunction of skin fibroblasts and epidermal keratinocytes and that this dysfunction is related to disrupted intracellular glutathione (GSH) homeostasis. METHODS: We investigated the effects of esterified GSH on the contraction of fibroblasts in a fibroblast-populated collagen lattice and on keratinocyte apoptosis. RESULTS: High glucose medium (hyperglycaemia) reduced the contraction ability of fibroblasts (P < 0.05). The normalization of glucose medium concentrations for hyperglycaemic fibroblasts did not restore the contraction capacity. The percentage of apoptotic keratinocytes was statistically higher in hyperglycaemic cells (P < 0.05). GSH media concentrations ranging from 0.1 to 100 micromol L(-1) restored the ability of hyperglycaemic fibroblasts to contract the gels in a concentration-dependent manner. Primary human keratinocytes grown in hyperglycaemic medium were more susceptible to apoptosis, and treatment with esterified GSH rescued the keratinocytes from apoptosis. CONCLUSIONS: These data suggest that intracellular GSH can normalize skin cell functions disrupted by in vitro cell growth under hyperglycaemic conditions.

The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes.

The effect of three different Korean Traditional Medicines (KTM) was studied on several functional parameters of adult human cells in culture. The cells were non-transformed strains of normal, skin epidermal cells (keratinocytes) from adult humans. Aqueous extracts of the herbal medicines were tested using two types of cell strains: one type was essential fatty acid deficient (EFAD) cells which grow rapidly in medium that was low in calcium and had no essential fatty acids; the second type was a cell strain grown in medium supplemented with essential fatty acid (EFA-supplemented). These cells had much slower, in vivo skin growth rates, and the fatty acid composition resembled that measured in epidermal biopsy tissue. The KTMs chosen for this study were tae-gang-hual-tang (for treating osteoarthritis), hual-ak-tang (for pain relief) and sip-zeon-tae-bo-tang (for fortifying immune systems). Because high proliferation rates usually correlate with skin inflammation and because many of the chemotactic agents mediating inflammatory response are modified fatty acids, this study focused on cell growth rate and membrane fatty acid composition as signals for the effects of the herbal medicines. By monitoring growth rate, these experiments measured both a stimulatory and a regulatory effect on the growth of keratinocytes. Some toxicity was seen at the highest doses of the KTMs. These effects were modeled mathematically, and the results showed varying effects on growth rate depending on dose and herbal recipe. The fitting parameters were discussed as they relate to biological function. The experimental design was also discussed and alternatives were suggested.

Human stratified squamous epithelia differ in cellular fatty acid composition.

The phospholipid component of the cellular membrane is crucial to the structure and function of cells. Basal cells from three epithelial tissues, adult human skin epidermis, oral mucosa, and hair follicles, grow rapidly in serum- and lipid-free medium. Analysis of phospholipid extracts from the above three types of stratified squamous epithelium in both in vivo and in vitro was done to relate fatty acid cell composition to cell function. The fatty acid composition of hair follicles in vivo was analyzed in plucked scalp hairs, and those of skin epidermis and oral mucosa in vivo were analyzed after separating the tissue into suprabasal and basal layers. The fatty acid composition of the in vivo cells from hair follicles shows a partial essential fatty acid (EFA)-deficient state. There was no significant difference between the skin epidermis and the oral mucosa in the fatty acid composition of the in vivo cells from each basal layer. However, in the suprabasal layers, the percent of linoleic acid (18:2) from the skin epidermis was higher than that from the oral mucosa. This study shows that total fatty acid composition in cell membranes of stratified squamous epithelium varies with their keratinization pattern. When cultured, the three types of rapidly growing keratinocytes showed the same essential fatty acid deficient pattern in the membrane phospholipids.

Development and characterization of a tissue-engineered human oral mucosa equivalent produced in a serum-free culture system.

A problem maxillofacial surgeons face is a lack of sufficient autogenous oral mucosa for reconstruction of the oral cavity. Split-thickness or oral mucosa grafts require more than one surgical procedure and can result in donor site morbidity. Skin has disadvantages of adnexal structures and a different keratinization pattern than oral mucosa. In this study, we successfully assembled, ex vivo, a human oral mucosa equivalent, consisting of epidermal and dermal components, in a defined, essential-fatty-acid-deficient, serum-free culture medium without a feeder layer, that could be used for intra-oral grafting in humans. Autogenous oral keratinocytes were seeded onto a cadaveric dermis, AlloDerm. The oral mucosa equivalent was cultured at an air-liquid interface for 2 wks. The resulting equivalent had a well-stratified parakeratinized epithelial layer similar to native oral keratinized mucosa. Expression of differentiation markers, filaggrin and cytokeratin 10/13, suggested a premature keratinized state. The presence of proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, suggested a state of hyperproliferation. Fatty acid composition of the equivalent was similar to that of in vitro cultured oral keratinocytes but differed from the that of in vivo native tissue, showing a lower content of 18:2 and 20:4, and a higher content of 16:1 and 18:1 fatty acids, respectively. The keratinocytes of the equivalent appeared to be in a more active and proliferative state than native keratinized mucosa. The dynamic nature of the cell population on the oral mucosa equivalent may be beneficial for intra-oral grafting procedures and for transfection of the keratinocytes.

Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes.

The fluorescent probe diI was used to study the lateral mobility of lipids in in vitro strains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 microM all-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement of D among the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lower D values. The other FRAP parameters (mobile fraction and bleach depth) were larger in the "slow" group than in the "fast" group. This trend of negative correlation between these parameters and D was also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.

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.

Substitution of D-Trp32 in NPY destabilizes the binding transition state to the Y1 receptor site in SK-N-MC cell membranes.

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.

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.

The effect of essential fatty acid supplementation on keratinocyte replication.

Epidermal cell growth in culture, using the low calcium, low serum technique described by Boyce, is thought to induce rapid expansion by inducing an essential fatty acid (EFA) deficiency state. To determine the mechanisms whereby EFA deficiency induces increased epidermal cell growth, keratinocytes were passaged into medium without or with the addition of EFAs, 18:2(n-6), 20:4(n-6). The resulting populations were assayed for replication rate, differentiation, and plating efficiency. Supplemental EFAs significantly decrease keratinocyte culture expansion. This is evidenced by an increase in generation time, a decrease in thymidine incorporation, and a decrease in modeled replication rate. EFA supplementation also increased the expression of cornified cell envelopes. Serum-free medium induces EFA deficient keratinocytes that demonstrate increased replication and decreased differentiation. Restoration of EFAs reverses these changes. It may be possible to manipulate keratinocyte physiology using fatty acid modifications.

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.

Fatty acid metabolism studies of human epidermal cell cultures.

Adult human epidermal keratinocytes grow rapidly in medium that is essential fatty acid (EFA)-deficient. In this medium they exhibit decreased amounts of the fatty acids, 18:2, 20:3, 20:4, and contain increased amounts of monounsaturated fatty acids. [14C]- and [3H]acetate and radiolabeled fatty acids, 16:0, 18:2, and 20:4 were used to study the fatty acid metabolism of these cells. Label from acetate appeared in 14- to 20-carbon fatty acids, both saturated and monounsaturated. No label was seen in the essential fatty acid 18:2, 18:3, and 20:4. Radiolabel from [9, 10-3H]palmitic acid (16:0) was detected in 16:0, 16:1, 18:0, and 18:1. [14C]linoleic acid (18:2) was converted to 18:3, 20:2, 20:3, and 20:4, demonstrating delta 6 and delta 5 desaturase activity in keratinocytes. Label from acetate, 16:0, or 18:2 was found mostly in the cellular phospholipids while only one third of the label from [14C]arachidonic was found in the phospholipids. [14C]acetate and [14C]18:2 time course data were used to construct a model of the metabolism of these reactants, using coupled, first-order differential equations. The data show that EFA-deficient keratinocytes metabolize fatty acids using pathways previously found in liver; they suggest the positioning of 18:2 desaturase and 18:3 elongase near the plasma membrane; they indicate that for the synthesis of nonessential fatty acids the formation of 18:0 from 16:0 is the rate-determining step; and they show that the conversion of 18:2 to 20:3 is rapid. These experiments demonstrate a method to study lipid enzyme kinetics in living cells.