Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Infaunal burrows are enrichment zones for Vibrio parahaemolyticus

Vibrio parahaemolyticus, a species that includes strains known to be pathogenic in humans, and other Vibrionaceae are common, naturally occurring bacteria in coastal environments. Understanding the ecology and transport of these organisms within estuarine systems is fundamental to predicting outbreaks of pathogenic strains. Infaunal burrows serve as conduits for increased transport of tidal waters and V. parahaemolyticus cells by providing large open channels from the sediment to salt marsh tidal creeks. An extensive seasonal study was conducted at the North Inlet Estuary in Georgetown, SC, to quantify Vibrionaceae and specifically V. parahaemolyticus bacteria in tidal water, fiddler crab (Uca pugilator, Uca pugnax) burrow water, and interstitial pore water. Numbers of V. parahaemolyticus bacteria were significantly higher within burrow waters (4,875 CFU ml(-1)) than in creek water (193 CFU ml(-1)) and interstitial pore water (128 CFU ml(-1)), demonstrating that infaunal burrows are sites of V. parahaemolyticus enrichment. A strong seasonal trend of increased abundances of Vibrionaceae and V. parahaemolyticus organisms during the warmer months of May through September was observed. Multilocus sequence typing (MLST) analysis of isolates presumed to be V. parahaemolyticus from creek water, pore water, and burrow water identified substantial strain-level genetic variability among V. parahaemolyticus bacteria. Analysis of carbon substrate utilization capabilities of organisms presumed to be V. parahaemolyticus also indicated physiological diversity within this clade, which helps to explain the broad distribution of these strains within the estuary. These burrows are "hot spots" of Vibrionaceae and V. parahaemolyticus cell numbers and strain diversity and represent an important microhabitat.     

Lipid alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

The lipid composition of cells of Pseudomonas aeruginosa strains resistant to polymyxin was compared with the lipid composition of cells of polymyxin-sensitive strains as to their content of readily extractable lipids (RELs), acid-extractable lipids, the fatty acid composition of RELs, and the contents of various phospholipids in the REL fraction. The polymyxin-resistant strains had an increased content of RELs, but a decreased phospholipid content. The REL fraction from the polymyxin-resistant strains had an increased content of unsaturated fatty acids accompanied by a decreased content of cyclopropane fatty acids as compared with the fatty acid composition of RELs from polymyxin-sensitive strains. The phosphatidylethanolamine content was greatly reduced in the polymyxin-resistant strains, whereas the content of an unidentified lipid, thought to be a neutral lipid lacking either a phosphate, free amino, or choline moiety, was greatly increased. Cell envelopes of the polymyxin-resistant strains contained reduced concentrations of Mg2+ and Ca2+ as compared with the cell envelopes of polymyxin-sensitive strains. It appears that polymyxin resistance in these strains is associated with a significant alteration in the lipid composition and divalent cation content of the cell envelope.     

Ultrastructural and Chemical Alteration of the Cell Envelope of Pseudomonas aeruginosa, Associated with Resistance to Ethylenediaminetetraacetate Resulting from Growth in a Mg2+-Deficient Medium

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.     

Analysis of the envelope proteins of heat-shocked Vibrio parahaemolyticus cells by immunoblotting and biotin-labeling methods

Vibrio parahaemolyticus, a common enteropathogen in tropical and subtropical coastal regions, exhibits significant adaptive acid tolerance response and heat-shock response, and the envelope proteins induced by stresses are suggested to be associated with virulence. This work examined the heat-shock proteins located in the envelope of V. parahaemolyticus by two rapid methods; namely, the immunoblotting and biotin-labeling methods. The bacterial cells were cultured at 25 C and heat shocked at 37 or 42 C for 1 or 2 hr. The cells were first lysed, then proteins were separated by gel electrophoresis and probed with antiserum raised against heat-shocked cells. Next, the heat-shocked cells were examined by labeling with water soluble sulfo-NHS-LC-biotin. Proteins of 33, 61, 66, 71, 78, 92 and 101 kDa were induced, while 55, 86, 102, 120 and 160 kDa proteins were markedly enhanced in the envelope of the heat-shocked V. parahaemolyticus cells. The biotin tagged envelope proteins were purified using a monomeric avidin column, and the N-terminal sequence was determined and compared with other high identity protein sequences. The sequence results suggest that Vph1 (55 kDa), Vph2 (46 kDa) and Vph3 (42 kDa) are de novo synthesized heat-shock proteins located in the envelope of this pathogen, and the functions of these proteins in stress protection and virulence have yet to be determined.     

Cell membrane stability and biochemical response of cultured cells of groundnut under polyethylene glycol-induced water stress

Cell membrane stability (CMS) in suspension cultures of two groundnut cultivars was studied under polyethylene glycol(PEG)-induced water stress. There was a negative relationship between PEG concentration in the medium and membrane stability measured as electrolyte leakage. The CMS values in the cell cultures correlated well with the whole plant tissue and permitted the differentiation of cultivars based on their known response to drought stress. The cell membrane stability was lower (more electrolyte leakage) when cells were grown in culture as compared to the intact plant tissue. Kadiri-3, the drought tolerant cultivar maintained higher CMS than JL-24, the drought susceptible one. With increasing PEG levels the concentration of Potassium in cultured cells declined in both cultivars. However, Kadiri-3 maintained higher K values than JL-24 accompanied with greater cell membrane stability. Total soluble sugars also increased with increasing stress in both cultivars; the increase being higher in Kadiri-3. There was no significant change in the total free amino acids but proline accumulated markedly in both varieties. However, no relationship was found between proline levels and CMS. The results demonstrated that CMS test can also be used under in vitro conditions to differentiate the drought tolerant and susceptible cultivars and the cellular K level has a positive relationship with membrane stability.     

Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli.

Supplementation of the growth medium with high concentrations of NaCl, KCl, or sucrose caused a drastic change in the ratio of the two peptidoglycan-associated major outer membrane proteins of Escherichia coli K-12 in that the amounts of proteins b and c present in cell envelope preparations decreased and increased, respectively. Kinetic studies showed that, after the osmolarity of the medium was changed, one protein was hardly incorporated into the membrane, whereas the other was incorporated with an increased rate. After about 1.5 to 2 generations, the cell envelopes obtained the b/c ratio characteristic for the new medium, and both proteins were subsequently incorporated in the cell ensured this new ratio. Once proteins b and c were incorporated in the cell envelope, they were not converted into each other by changes in osmolarity of the growth medium.     

Effects of reduction of auxin and destruction of microtubules on cell wall proteins and cell morphology of the BY-2 line of tobacco cells

Variations of cell wall proteins and proteins in the medium associated with changes in cell morphology were investigated in the BY-2 line of cultured cells. BY-2 cells cultured in LS medium grew as long chains of cells, with the plane of division perpendicular to the longitudinal axis. Reduction in the levels of auxin in the medium resulted in inhibition of cell division and promotion of cell elongation. Levels of cell wall proteins in cell walls decreased and relative levels of cell wall proteins and proteins in the medium changed. Upon treatment with the anti-microtubule drug, propyzamide, cells expanded laterally. Level of cell wall proteins and relative levels of individual cell wall proteins did not change very much, but levels of proteins in the culture medium increased. In both cases, levels of acid and basic peroxidases in cell walls increased and isozyme patterns of these changed.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Physiological Changes in Rhizobia after Growth in Peat Extract May Be Related to Improved Desiccation Tolerance

Improved survival of peat-cultured rhizobia compared to survival of liquid-cultured cells has been attributed to cellular adaptations during solid-state fermentation in moist peat. We have observed improved desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 and Bradyrhizobium japonicum CB1809 after aerobic growth in water extracts of peat. Survival of TA1 grown in crude peat extract was 18-fold greater than that of cells grown in a defined liquid medium but was diminished when cells were grown in different-sized colloidal fractions of peat extract. Survival of CB1809 was generally better when grown in crude peat extract than in the control but was not statistically significant (P > 0.05) and was strongly dependent on peat extract concentration. Accumulation of intracellular trehalose by both TA1 and CB1809 was higher after growth in peat extract than in the defined medium control. Cells grown in water extracts of peat exhibit morphological changes similar to those observed after growth in moist peat. Electron microscopy revealed thickened plasma membranes, with an electron-dense material occupying the periplasmic space in both TA1 and CB1809. Growth in peat extract also resulted in changes to polypeptide expression in both strains, and peptide analysis by liquid chromatography-mass spectrometry indicated increased expression of stress response proteins. Our results suggest that increased capacity for desiccation tolerance in rhizobia is multifactorial, involving the accumulation of trehalose together with increased expression of proteins involved in protection of the cell envelope, repair of DNA damage, oxidative stress responses, and maintenance of stability and integrity of proteins.     

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane.     

Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268

Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein. A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S. ruminantium OB268 showed that they consisted primarily of the 42 kDa protein. Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S. ruminantium D1 and S. infelix, indicating a conservation of antigenic structure among each of the major envelope proteins. The N-terminus of the 42 kDa S. ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain. Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer. These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein. The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.     

Cloning and sequencing of the gene encoding an aldehyde dehydrogenase that is induced by growing Alteromonas sp. Strain KE10 in a low concentration of organic nutrients

The protein composition of Alteromonas sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter(-1)) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD(+)-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in Escherichia coli, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.     

Regulation of lipid accumulation in 3T3-L1 cells: insulin-independent and combined effects of fatty acids and insulin

The insulin-independent and combined effects of fatty acids (FA; linoleic and oleic acids) and insulin in modulating lipid accumulation and adipogenesis in 3T3-L1 cells was investigated using a novel protocol avoiding the effects of a complex hormone 'induction' mixture. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus serum (control) or in DMEM plus either 0.3 mmol/l linoleic or oleic acids with 0.3 mmol/l FA-free bovine serum albumin in the presence or absence of insulin. Cells were cultured for 4 to 8 days and cell number, lipid accumulation, peroxisome proliferator-activated receptor-gamma (PPAR-γ) and glucose transporter 4 (GLUT-4) protein expression were determined. Cell number appeared to be decreased in comparison with control cultures. In both oleic acid and linoleic acid-treated cells, notably in the absence (and presence) of insulin, oil-red O stain-positive cells showed abundant lipid. The percentage of cells showing lipid accumulation was greater in FA-treated cultures compared with control cells grown in DMEM plus serum (P < 0.001). Treatment with both linoleic and oleic acid-containing media evoked higher levels of PPAR-γ than observed in control cultures (P < 0.05). GLUT-4 protein also increased in response to treatment with both linoleic and oleic acid-containing media (P < 0.001). Lipid accumulation in 3T3-L1 cells occurs in response to either oleic or linoleic acids independently of the presence of insulin. Both PPAR-γ and GLUT-4 protein expression were stimulated. Both proteins are considered markers of adipogenesis, and these observations suggest that these cells had entered the physiological state broadly accepted as differentiated. Furthermore, 3T3-L1 cells can be induced to accumulate lipid in a serum-free medium supplemented with FA, without the use of induction protocols using complex hormone mixtures. We have demonstrated a novel model for the study of lipid accumulation that will improve the understanding of adipogenesis in adipocyte lineage cells.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

A Morphometric Study of Euryhalinity in Marine Populations of the Ciliate Genus Euplotes

ABSTRACT. Twenty different clonal strains of marine and brackish Euplotes , representing four morphotypes, were tested for hyposalinity tolerance by a method which gradually acclimated the cells to lower salinity medium. The lowest salinities in which the strains could thrive ranged from 60% of normal seawater to complete freshwater. The morphological effects of culture medium salinity were also examined for two strains of a small "Euplotes charon" morphotype, as well as for two mating compatible "Euplotes vannus" strains and several of their exconjugates. There were no differences between the euryhaline strains grown in fresh or saltwater, except for a slight increase in overall cell size in one strain when cultured in freshwater medium. E. vannus strains increased in overall cell size with decreased salinity; also, the dorsal surface of the cells can become disorganized when the cells are cultured in 30% normal seawater.     

Characteristics of Escherichia coli grown in bay water as compared with rich medium

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

Summary Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes. This work was supported in part by NATO Scientific Award (RG 8510056).