Accurate and Efficient One-Pot Reverse Transcription and Amplification of 2'-Fluoro-Modified Nucleic Acids by Commercial DNA Polymerases.

DNA is a foundational tool in biotechnology and synthetic biology but is limited by sensitivity to DNA-modifying enzymes. Recently, researchers have identified DNA polymerases that can enzymatically synthesize long oligonucleotides of modified DNA (M-DNA) that are resistant to DNA-modifying enzymes. Most applications require M-DNA to be reverse transcribed, typically using a RNA reverse transcriptase, back into natural DNA for sequence analysis or further manipulation. Here, we tested commercially available DNA-dependent DNA polymerases for their ability to reverse transcribe and amplify M-DNA in a one-pot reaction. Three of the six polymerases chosen (Phusion, Q5, and Deep Vent) could reverse transcribe and amplify synthetic 2'F M-DNA in a single reaction with <5 × 10-3 error per base pair. We further used Q5 DNA polymerase to reverse transcribe and amplify M-DNA synthesized by two candidate M-DNA polymerases (SFP1 and SFM4-6), allowing for quantification of the frequency, types, and locations of errors made during M-DNA synthesis. From these studies, we identify SFP1 as one of the most accurate M-DNA polymerases identified to date. Collectively, these studies establish a simple, robust method for the conversion of 2'F M-DNA to DNA in <1 h using commercially available materials, significantly improving the ease of use of M-DNA.

Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation.

Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.

Ceramides are transported through the Golgi apparatus in human keratinocytes in vitro.

The intercellular lipid sheets of the stratum corneum constitute the epidermal permeability barrier that permits terrestrial life. Although lamellar granules are known to deliver the precursors of the stratum corneum lipids into the intercellular spaces, their site of origin remains unknown. Lamellar granules have characteristics of both secretory granules and lysosomes, which are known to originate from the Golgi apparatus in other cell types. Glucosylceramides, a major component of lamellar granule contents and the precursors of stratum corneum ceramides, have been found to be synthesized primarily in the early compartments of the Golgi apparatus in other cell types. We have investigated the transport and metabolism of a fluorescently labeled ceramide in human keratinocyte cultures using laser-scanning confocal microscopy and lipid analysis. We found that ceramide is metabolized to glucosylceramide and sphingomyelin as it passes through the Golgi apparatus and the metabolites are then delivered to the plasma membrane. Cold temperature, Brefeldin A, and monensin, all known to inhibit transport from the Golgi to the plasma membrane, prevented ceramide metabolites from appearing at the plasma membrane. Because glucosylceramides are one of the most important lipid constituents of lamellar granules, these results support the hypothesis that the Golgi is the origin of lamellar granules.

Covalently bound lipids of human stratum corneum.

In the present study, we demonstrate that human stratum corneum contains covalently bound lipids accounting for 1.4% of the dry weight of the tissue. The major component (53.3% of the total by weight) is a ceramide (CER-A) consisting of 30 through 34-carbon omega-hydroxyacids amide-linked to sphingosine. The other bound lipids in human stratum corneum include fatty acids (12.7%), omega-hydroxy acids (9.4%), and a second, more polar, omega-hydroxyacid-containing ceramide (CER-B, 24.8%). The predominant omega-hydroxyacids in both ceramides, as well as the free hydroxyacid fraction, are the 30-carbon saturated and 32- and 34-carbon monoenoic species. The bound fatty acids consist largely of 14 through 22-carbon saturated species, but significant proportions of monoenoic species and linoleic acid are also present. Psoriatic scale contains a similar total concentration of the same covalently bound lipids, but the proportions of the individual bound lipids are different from those found in normal stratum corneum. It is suggested that the principal function of the covalently bound lipids in human stratum corneum is the formation of a lipid envelope on the outer surface of the keratinized cells.

Sphingolipid metabolism in organotypic mouse keratinocyte cultures.

Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine. These results indicate that considerable diversity of ceramide structures occurs among mammalian species and that cultured keratinocytes may only partially reproduce the in vivo complement of ceramides. Using labeled serine in keratinocyte cultures, we have also demonstrated the de novo synthesis of ceramides and the transfer of label from glucosylceramides to ceramides during terminal differentiation of lifted cultures. The covalently bound corneocyte lipid envelope, which has recently been characterized in pig and human epidermis, was also present in mouse epidermis and was reproduced by the lifted cultures. Very-long-chain omega-hydroxyceramides were the dominant bound lipid and labeling studies in culture indicated that they were derived from ceramides synthesized in the viable epidermis.

Lipid composition of cultured murine keratinocytes.

The current study was undertaken to determine whether a previously reported murine culture system is an acceptable model for the study of epidermal lipid metabolism. The lipid composition of primary neonatal mouse keratinocyte cultures was determined and compared with that of freshly isolated keratinocytes and whole epidermis. 14C-Labeled arachidonic acid (AA) and linoleic acid (LA) were added to cultures and the incorporation into specific lipids was assessed. The lipid composition of the cultures indicated that they were partially differentiated, which parallels the well-known incomplete keratinization seen in many keratinocyte culture systems. Of particular importance, the LA-rich uniquely epidermal lipids which may be of importance in water barrier function, acylglucosylceramide (AGC) and acylceramide (AC), were made by the cultures. Fatty acid analysis of total lipid, phospholipid, and AGC extracts revealed a significant decrease in LA content compared with the parent epidermis; this may have resulted from the low level of LA in fetal bovine serum, which was the serum source for these cultures. Labeled AA and LA were incorporated into the lipids of cultured keratinocytes in distinct patterns that were consistent with the fatty acid content of the lipids. Both AGC and AC showed preferential uptake of LA compared with AA. There was minimal labeling of non-linoleate-containing lipids and a low degree of conversion of labeled LA to AA. Considering the grossly different environment of the in vitro system compared with the in vivo state, the overall lipid composition was remarkably well maintained. Keratinocyte cultures should be of great value in the study of epidermal lipid metabolism.

The role of the corneocyte lipid envelopes in cohesion of the stratum corneum.

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45 degrees C without agitation. This is prevented by prior heating of the tissue to 80 degrees C or by solvent extraction of the intercellular lipids. In the present study, electron microscopy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the omega-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitation of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of intercellular lamellae allows the appropriate cell-to-cell contact.

Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum.

The epidermal permeability barrier necessary for terrestrial life resides in the intercellular spaces of the stratum corneum and is composed of lipids. Membrane coating granules (MCGs), small intracellular organelles found in the uppermost layers of the living epidermis, contain stacks of membranous disks which are extruded into the intercellular space and undergo both biochemical and physical changes to form the lipid sheets which constitute this barrier. Using ruthenium tetroxide as a secondary fixative, we are able to demonstrate stacks of lamellae filling the intercellular spaces in the uppermost layers of the stratum corneum. The structure of these lipid lamellae is consistent with the proposed derivation of MCG lipid disks and also suggests that the lipid bilayer adjacent to the corneocyte cell envelope may be assembled from lipids not derived from MCGs.

Evidence that the corneocyte has a chemically bound lipid envelope.

The stratum corneum of mammalian epidermis contains a mixture of ceramides, free fatty acids, cholesterol, and cholesteryl sulfate, amounting to 14% of the dry weight of the tissue, that can be removed by exhaustive extraction with chloroform/methanol. Subsequent mild alkaline hydrolysis liberates additional lipid, consisting almost exclusively of C30-C34 omega-hydroxyacids in amide linkage with sphingosine, equal to 2% of the tissue mass. In the present study, transmission electron microscopy was used to demonstrate that the initial extraction removes the intercellular lamellae that constitute the epidermal water barrier but leaves the lucent band that has been termed the corneocyte plasma membrane. The subsequent alkaline hydrolysis and lipid extraction remove the lucent band, which must therefore contain the omega-hydroxyacylsphingosines. From the results of in situ derivatization of these lipids and the construction of molecular models, it is inferred that the bound lipids exist in ester linkage with protein on the surface of the corneocyte envelope. The tightly packed hydroxyacylsphingosine molecules thus form a lipid envelope for each corneocyte.

Composition and morphology of epidermal cyst lipids.

The contents of epidermal cysts were used as a source of desquamated human keratinocytes uncontaminated by sebaceous, subcutaneous, or bacterial lipids. Lipids extracted with chloroform:methanol mixtures included six series of ceramides (41% of the total extractable lipid), cholesterol (27%), cholesteryl esters (10%), fatty acids (9%), cholesteryl sulfate (1.9%), a novel class of ceramide esters (3.8%), and a sterol diester (0.9%). Electron microscopy revealed that the lipids in the cyst contents existed as multiple intercellular lamellae, as in stratum corneum. One lamella, adjacent to the horny cell protein envelope, was resistant to lipid extraction and is thought to represent covalently bound lipid on the outer surface of the keratinocyte. The results indicate that the degradation of intercellular lipid lamellae is not required for desquamation.

Lamellar granule extrusion and stratum corneum intercellular lamellae in murine keratinocyte cultures.

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shows morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30 mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 micrograms/ml each of insulin and hydrocortisone, and kept at 32 degrees C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated.

Fatty acids of acylceramides from comedones and from the skin surface of acne patients and control subjects.

Comedonal lipids and skin surface lipids were collected from six acne patients and surface lipids were collected from sex- and age-matched controls without acne. Six series of ceramides were found in each sample, the relative amounts of which were determined by thin-layer chromatography/photodensitometry. Acylceramides (ceramide 1) were isolated by preparative thin-layer chromatography and their ester-linked fatty acids were analyzed by gas-liquid chromatography. The comedonal acylceramides contained higher proportions of 16:0, 16:1 delta 6, and 18:1 delta 6 + delta 8 and much less linoleate (18:2 delta 9,12) than the acylceramides from the skin surface. In the surface lipids from legs, acylceramides from the acne patients contained less linoleate than the acylceramides from control subjects. Free fatty acids from the comedones were also isolated and analyzed, and had a composition very similar to the esterified fatty acids of comedonal acylceramides. The results confirm that fatty acids derived from sebum become incorporated into comedonal acylceramides, displacing linoleate, and show that this process even affects the acylceramides of surface epidermis, more so in acne patients than in normal subjects.

Murine keratinocyte cultures grown at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during differentiation.

In a recent investigation we showed that murine keratinocyte cultures grown at the air/medium interface in the presence of dermis exhibit morphologic differentiation comparable to that seen in vivo, including the formation of lamellar granules and stratum corneum intercellular lipid lamellae. In the present study, lifted cultures were found to more closely reproduce the lipid composition of the parent epidermal tissue than submerged cultures grown on plastic. In addition, the specific fatty acid profile of individual lipid classes in lifted cultures was, in general, remarkably well maintained in vitro. Acylceramides, which are highly enriched in linoleic acid in vivo, remained enriched in vitro; however, the linoleic acid content of the cultures was substantially lower than that in vivo, confirming previous reports of the relative essential fatty acid deficiency of standard culture media. As the lifted cultures differentiated over time, the lipid composition changed to reflect the formation of a stratum corneum with its different complement of lipids. Label from [U-14C]linoleic acid was specifically incorporated into linoleate-containing lipids during short pulses in both submerged and lifted cultures. Changes in label distribution over a long chase period in lifted cultures indicated that linoleate was transferred from phospholipids to ceramides, providing evidence for the "recycling" of essential fatty acids in epidermis.

Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.

Intercellular lipid lamellae in the stratum corneum constitute the barrier to water diffusion and may also play a role in cohesion between corneocytes. The lamellae arise from stacks of lamellar disks that are extruded from the granular cells and then fuse edge-to-edge to form sheets. It has been proposed that each lamellar disk is formed from a flattened vesicle, and therefore consists of two lipid bilayers in close apposition. In the present study, electron microscopic examination of ruthenium-tetroxide-fixed stratum corneum from mouse, pig, and human skin revealed that the double bilayer pattern persists in the intercellular lamellae. In addition, distinctive patterning of the intercellular lamellae has led us to propose novel molecular arrangements of the intercellular lipids. These include interlamellar sharing of lipid chains to produce lipid monolayers between pairs of bilayers. The pattern reflects the provenance of the intercellular lamellae from lamellar granule disks and the nonrandom orientation of the lamellar lipids.

Osmium tetroxide and ruthenium tetroxide are complementary reagents for the preparation of epidermal samples for transmission electron microscopy.

Ruthenium tetroxide and osmium tetroxide were compared as post-fixatives in the preparation of human epidermis for transmission electron microscopic examination. Both reagents revealed characteristic lamellar granules within the granular layer and extruded lamellar granule contents in the upper granular layer. The transformation of the granule contents into multilamellar sheets at the interface between the granular and cornified layers and the persistence of these sheets through all levels of the stratum corneum were demonstrated only with ruthenium tetroxide fixation. Therefore, the reactivity of osmium tetroxide with isolated epidermal lipids was examined. The failure of osmium tetroxide to reveal membrane structures in the stratum corneum can be explained by its inability to react with many of the lipid components of these membranes, rather than to selective removal of lipids during tissue processing, as was formerly believed. Ruthenium tetroxide, a stronger oxidizing agent than osmium tetroxide, overcomes this problem but has other severe limitations as a post-fixative.

Caveolin expression and localization in human keratinocytes suggest a role in lamellar granule biogenesis.

Lamellar granules are sphingolipid-enriched organelles, probably intimately related to the tubulo-vesicular elements of the trans-Golgi network, that deliver the precursors of stratum corneum barrier lipids to the extracellular compartment. Caveolins are cholesterol-binding scaffolding proteins that facilitate the assembly of cholesterol- and sphingolipid-enriched membrane domains known as caveolae. Similarities in the composition of lamellar granules and caveolae suggest that caveolins could be involved in lamellar granule assembly, trafficking, and/or function. In order to explore this relationship, we have examined the expression of caveolins in epidermis, keratinocyte cultures, and an isolated lamellar granule fraction using immunolabeling, immunoblotting, and northern blotting. Several antibodies show immunolocalization of caveolin-1 in the basal layer of human epidermis, with a decline in the suprabasal layers and a reemergence of expression at the stratum granulosum/stratum corneum junction. Two of three caveolin-2 antibodies show little basal staining, but strong signal throughout the rest of the epidermis, whereas a third shows a pattern like caveolin-1. An antibody against caveolin-3 shows a strong signal at the stratum granulosum/stratum corneum interface. Caveolins partially colocalize with glucocerebrosidase, an enzyme known to be critical for remodeling of extruded lamellar granule contents, with AE17, a previously described lamellar-granule-associated antibody, and with glucosylceramides, a major lipid component of lamellar granules. Caveolin-1 protein is present in undifferentiated low-calcium-grown keratinocyte cultures, decreases upon induction of differentiation, and then rises to levels above those seen in undifferentiated cultures, consistent with the immunofluorescence findings. Caveolin-1 mRNA expression parallels that of the protein. Caveolin-2 mRNA and protein expression were unchanged over the course of culture differentiation. Keratinocyte caveolin-1 mRNA expression is not induced by an increase in medium calcium level and is markedly reduced by phorbol-ester-mediated protein kinase C induction. Caveolin-1 is enriched in an isolated lamellar granule fraction that is also enriched, as we have previously described, in lysosomal acid lipase and glucocerebrosidase, and localizes to structures consistent with lamellar granules on immunoelectron microscopy. The differentiation-dependent expression of caveolin-1, the colocalization of caveolins with putative lamellar-granule-associated antigens, their enrichment in isolated lamellar granules, and their presence in lamellar-granule-like structures on immunoelectron microscopy, along with their known structural role in the assembly of glycosphingolipid- and cholesterol-enriched domains in other cell types, suggest that caveolins may play a role in lamellar granule assembly, trafficking, and/or function.

Essential fatty acids and epidermal integrity.

The intercellular spaces of the stratum corneum contain multilamellar lipid sheets derived from the extruded contents of lamellar granules. In the absence of linoleic acid, lamellar granules appear empty, and only fragmentary extracellular sheets are found. This defective differentiation is attributable to substitution of oleate for linoleate in O-acylsphingolipids. Normally, linoleate is ester-linked to 30- to 34-carbon omega-hydroxyacids, which, in turn, are amide-linked to sphingosine. Acylglucosylceramides, bearing a beta-D-glucosyl moiety on the sphingosine, may provide the driving force for lamellar granule assembly. The omega-hydroxyacyl chains are long enough to span a lipid bilayer, while the linoleate inserts into an adjacent bilayer. This interaction could promote assembly of lamellar granules. It has also been proposed that acylceramides may stabilize the extracellular sheets by a similar mechanism. In addition, the horny cell has been found to possess a covalently bound lipid envelope consisting principally of omega-hydroxyacylsphingosines derived from O-acylsphingolipids.

Regulation of the expression of epidermal keratinocyte proliferation and differentiation by vitamin A analogs.

A number of vitamin A analogs (retinoids) were used to manipulate the growth of epidermal keratinocytes in culture. The retinoids used were the TMMP analog of ethyl retinoate (Ro 10-9359), 13-cis retinoic acid, all trans retinoic acid and retinol (trans). These were added to primarily neonatal mouse epidermal keratinocyte cultures that proliferate, stratify, and differentiate over 2-3 weeks. [3H]Tdr labeling technics were used to quantitate proliferation. A histologic stain, and a four buffer protein extraction protocol, used in conjunction with polyacrylamide gel electrophoresis and fluorographic technics, were used to assess the differentiation of the cultures. Our results showed that all of the vitamin A analogs we tested inhibited keratinocyte proliferation. Quantitation of specific differentiation proteins showed that Ro 10-9359 and 13-cis retinoic acid partially inhibited the differentiation of the cultures. The Ro 10-9359 retinoid was unusual in that it increased the synthesis of keratohyalin granule-related proteins. These studies showed that inhibition of basal cell proliferation did not result in the obligatory expression of cell differentiation and that at least one of the events that is a part of epidermal keratinocyte differentiation can be separately controlled.

Isolation of corneocyte envelopes from porcine epidermis.

Sheets of porcine stratum corneum were dispersed into individual corneocytes after 4 h in a solution consisting of 8 mM N,N-dimethyldodecylamine oxide and 2 mM sodium dodecylsulfate in phosphate-buffered isotonic saline, at 45 degrees C. With continued detergent treatment and moderate sonication, most of the cells lost their keratin contents and were then separated from the remaining intact cells by centrifugation in cesium chloride solution of density 1.280. Electron microscopy showed that the cell envelopes retained both the crosslinked protein envelope and its attached lipid envelope. The dry weight of envelopes was approximately 7% of the estimated dry weight of the original stratum corneum, while the corneocytes surviving intact also amounted to 7% of the starting weight. Mild alkaline hydrolysis of the corneocyte envelopes allowed the extraction of hydroxyceramides amounting to 10% of the dry weight of the envelopes. The procedure therefore provides isolated corneocyte envelopes suitable for studying both the protein and lipid components of this compound sheath.

Miliaria crystallina occurring in a patient treated with isotretinoin.

A case of miliaria crystallina occurring with isotretinoin therapy in a patient with lamellar ichthyosis is described. To our knowledge, the association of miliaria crystallina with isotretinoin therapy has not been previously reported.