CELL MEMBRANE COMPLEX 445 Furthermore, evidence has been provided confi rming the existence of cholesterol sulfate in human hair by Wertz and Downing (34) and by Korner et al. in wool fi ber (48). The work of Korner et al. (48) builds upon the fi ndings of Wertz et al. on liposome forma- tion and lipids from stratum corneum (47), where Korner et al. (48) demonstrated that cell membrane lipids extracted from human hair and wool fi ber with chloroform/metha- nol/aqueous potassium chloride can form liposomes, providing evidence for a bilayer structure of the internal lipids of the beta layers of the cortical CMC in wool fi ber and in human hair (see Figure 3). Such extracts must come primarily from the cortex–cortex CMC because covalently bound MEA and the other covalently bound lipids of the cuticle CMC are not removed with this solvent system. Therefore, if the beta layers of the cuticle cells are primarily covalently bound fatty acids with some free lipids (see Figure 2) and the beta layers of cortical cells consist primarily of lipid bilayers (Figure 3), then it is highly likely that the proteins that these very differ- ent lipid layers are attached to, which are the cell membrane proteins, and the delta layer proteins of the cuticle cells and cortical cells are also different (see the section entitled “Proteins of the CMC” and the next part of this section). Additional differences between cuticle–cuticle, cuticle–cortex, and cortex–cortex CMC. As early as 1975, Nakamura et al. (8) provided evidence by staining reactions that the disulfi de con- tent in the delta layer in cuticle–cuticle CMC is lower than the disulfi de content of the delta layer in either cuticle–cortex or cortex–cortex CMC and that the carboxyl content in the cuticle–cuticle CMC is higher than that of the cortex–cortex CMC. In addition, Nakamura et al. added that the delta layer of the cuticle–cuticle CMC stains similar to the endocuticle. In 1983, Leeder et al. (44) in a TEM study involving the effect of solvents on wool fi bers found that formic acid treatment of wool fi ber modifi ed the CMC of the fi bers, but this effect was only observed between adjacent cortical cells and not between cuticle and corti- cal cells. These scientists suggested that these results are consistent with differences in the CMC between cuticle cells versus the CMC between cuticle and cortical cells. Peters and Bradbury (49) observed by electron microscopy that formic acid treatment of wool modifi ed the cell membrane complex of the cortex, “but that of the cuticle ap- pears unchanged.” They also analyzed “resistant membranes” from cuticle and cortical cells isolated by shaking wool fi bers in formic acid and oxidized with performic acid, and found differences, although their results for the Allworden membrane provided considerably lower values for cystine than the analysis of Allworden membranes by Allen et al. (30). Peters and Bradbury concluded that the “CMC of the cuticle differs from that of the cortex.” Leeder et al. in 1985 (50) described differences in the staining characteristics of the cuticle– cuticle CMC, the cuticle–cortex CMC, and the cortex–cortex CMC. After dyeing the fi bers with a uranyl dye, these scientists found a layer of dye around each cuticle cell, which was restricted to the CMC of the cuticle and not in the CMC of the cortex. They found only one dye layer at the cuticle–cortex junction and none in the cortex–cortex CMC, but two layers of dye in the cuticle–cuticle CMC, and they referred to the observa- tions of Nakamura et al. (8) on differences in the staining characteristics of these three types of CMC. Mansour and Jones, in 1989 (37), treated wool by Soxhlet extraction with chloroform/ methanol for fi ve hours, and subsequently in boiling water for 15 minutes, and examined

JOURNAL OF COSMETIC SCIENCE 446 the fi bers by electron microscopy after each stage of treatment. After the initial solvent ex- traction, the cuticle–cortex CMC appeared unmodifi ed, while the staining intensity of the beta layers between cortical cells were changed and appeared “intermittent.” After solvent extraction for fi ve hours and hydrolysis for 15 minutes, signifi cant structural changes were observed. The cortex–cortex CMC showed an overall reduction in defi nition in the delta layer, and the beta layers displayed a lack of clear defi nition. These scientists suggested that solvent extraction of intercellular lipids makes the hair more vulnerable to hydrolytic dam- age, with the largest changes in the CMC occurring in the cortex–cortex CMC, which these scientists believe is related to a reduction in tear strength of wool fi ber by solvent extraction and hydrolysis. These results show that the cuticle–cortex CMC behaves differently from the cortex–cortex CMC in reaction to solvent extraction. The cuticle–cortex CMC is damaged by solvent extraction and subsequent hydrolysis, but not as severely as the cortex–cortex CMC. Logan et al., in 1990 (38), examined wool fi bers by TEM after extraction with chloro- form/methanol and found that the cuticle–cuticle CMC appeared unchanged compared to untreated fi bers. On the other hand, they found that the delta layer in the cortex was smaller and displayed variable staining intensity in most regions, which they deduced as “incomplete or preferential extraction.” These scientists examined fi ber sections after chloroform/methanol extraction, followed by treatment with formic acid, and noted large changes in the beta and delta layers of the cortex–cortex CMC that were “rarely observed” in the cuticle–cuticle CMC. They concluded that these results show that “inherent differ- ences exist between CMC’s of cuticle and those of cortical cells.” Negri et al. (39) in a 1996 paper referred to the work of Leeder et al. (50) and cited the work of Leeder, Bishop, and Jones (44), who showed that the unstained beta layers of the cuticle and cortex react differently to formic acid treatment. Formic acid removes pro- teins (51) from the cortex–cortex CMC (40), and it modifi es the CMC junctions of the cortex but not the cuticle–cuticle CMC junctions they referenced Nakamura (8), Leeder, Bishop, and Jones (44), and Peters and Bradbury (49) on these effects. They concluded that these observations suggest that only the beta layers of the cuticle–cuticle CMC con- tain covalently bound lipids, while the beta layers of the cortex contain lipids and a “stain-resistant membrane protein” that is “likely to be of a different structure than the cuticle membrane.” Inoue et al. in 2007 (52), by microbeam X-ray diffraction described that extraction of human hair with polar organic solvents (methanol or chloroform/methanol) at 37 degrees C for six hours removed some material from the delta layer of the cuticle–cuticle CMC, but that the beta layers were unaffected. On the other hand, the beta layers of the cuticle– cuticle CMC appeared to be affected by hexane extraction under the same conditions. The observation that changes in the delta layer of the cuticle–cuticle CMC by chloroform/ methanol extraction could be detected suggests that this method is more sensitive than TEM (38). The fact that Inoue et al. observed changes in the beta layers of the cuticle– cuticle CMC by hexane extraction could result from removal of free lipids between the covalently bound fatty acids of the cuticle–cuticle CMC (Figure 2), resulting in the fold- ing back of the covalently bound fatty acids in the beta layers and accounting for the differences found. The above discussion shows clearly that both the lipid beta layers and the proteins of the cell membranes and those of the delta layer of the cuticle–cuticle CMC differ from those of the cortex–cortex CMC, with evidence for differences from the cuticle–cortex CMC also.