Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression

We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.     

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes

The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent "permanent" markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentiation. Immunofluorescence staining data suggested that the antibodies were specific for keratin-type intermediate filaments. The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot technique. AE1 reacted with 56 and 50 kdalton keratins, AE2 with 65-67 and 56-kdalton keratins, and AE3 with 65-67 and 58 kdalton keratins. Thus all major epidermal keratins were recognized by at least one of the monoclonal antibodies. Moreover, common antigenic determinants were present in subsets of epidermal keratins. To correlate the expression of specific keratins with different stages of in vivo epidermal differentiation, the antibodies were used for immunohistochemical staining of frozen skin sections. AE1 reacted with epidermal basal cells, AE2 with cells above the basal layer, and AE3 with the entire epidermis. The observation that AE1 and AE2 antibodies (which recognized a common 56 kdalton keratin) stained mutually exclusive parts of the epidermis suggested that certain keratin antigens must be masked in situ. This was shown to be the case by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique. The results from these immunohistochemical and biochemical approaches suggested that: (a) the 65- to 67-kdalton keratins were present only in cells above the basal layer, (b) the 58-kdalton keratin was detected throughout the entire epidermis including the basal layer, (c) the 56- kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer, and (d) the 50-kdalton keratin was the only other major keratin detected in the basal layer and was normally eliminated during s. corneum formation. The 56 and 65-67- kdalton keratins, which are characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization.     

Expression of epidermal keratins and filaggrin during human fetal skin development

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins

Recent data have indicated that specific keratin molecules can provide useful markers for studying different types and stages of epithelial differentiation. To utilize these protein markers, however, it is important to establish the keratin nature of the molecules and identify unambiguously the individual keratin species. In this paper, we show that this can be done relatively easily by one- and two-dimensional gel electrophoresis combined with immunoblotting using three monoclonal antibodies (aIF, AE1, and AE3). The aIF antibody has previously been shown to crossreact with all classes of intermediate-filament proteins. Using one- and two-dimensional immunoblotting, we establish that this antibody recognizes all known epithelial keratins of human and rabbit, although the reaction is relatively strong for the larger, basic keratins and is relatively weak for some of the smaller, acidic keratins. In contrast, AE1 and AE3 monoclonal antibodies have previously been shown to be highly specific for the acidic and basic subfamilies of the keratins, respectively. The combined use of the broadly reacting aIF antibody and the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies should facilitate the immunological definition, identification, and classification of mammalian epithelial keratins.     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co- expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.     

The human thymic microenvironment: Thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and characterization of keratins in the epidermis of the tuatara (Sphenodon punctatus; Lepidosauria, Reptilia)

Reptilian scales are mainly composed of alpha-and beta-keratins. Epidermis and molts from adult individuals of an ancient reptilian species, the tuatara (Sphenodon punctatus), were analysed by immunocytochemistry, mono- and bi-dimensional electrophoresis, and western blotting for alpha- and beta-keratins. The epidermis of this reptilian species with primitive anatomical traits should represent one of the more ancient amniotic epidermises available. Soft keratins (AE1- and AE3-positive) of 40-63 kDa and with isoelectric points (pI) at 4.0-6.8 were found in molts. The AE3 antibody was diffusely localised over the tonofilaments of keratinocytes. The lack of basic cytokeratins may be due to keratin alteration in molts, following corneification or enzymatic degradation of keratins. Hard (beta-) keratins of 16-18 kDa and pI at 6.8, 8.0, and 9.2 were identified using a beta-1 antibody produced against chick scale beta-keratin. The antibody also labeled filaments of beta-cells and of the mature, compact beta-layer. We have shown that beta-keratins in the tuatara resemble those of lizards and snakes, and that they are mainly basic proteins. These proteins replace cytokeratins in the pre-corneoum beta-layers, from which a hard, mechanically resistant corneoum layer is formed over scales. Beta-keratins may have both a fibrous and a matrix role in forming the hard texture of corneoum scales in this ancient species, as well as in more recently evolved reptiles.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

Keratin protein expression during the development of conducting airway epithelium in nonhuman primates

Keratin protein expression during the development of Rhesus monkey conducting airway epithelium was investigated by both biochemical and immunohistochemical methods. Keratin proteins were extracted from tracheal and intrapulmonary airway tissues of fetal (at 80- and 140-day gestational ages), neonatal, and adult animals. Using immunoblot analyses and immunohistochemistry with various monoclonal (AE1, AE3, AE8, 6.01 and 6.11) and monospecific antibodies (anti-50/55 and anti-40 kDa), the presence of keratins 5, 6, 8, 13, 14, and 19 in adult airway epithelium were demonstrated. Except for keratin 13 (51 kDa), the remaining keratins could be immunologically detected in fetal and neonatal tissues. To further understand the nature of the synthesis of keratin 13 during development, airway epithelial cells from different ages were isolated and cultured in vitro. Cultured cells were labeled with 35S-methionine, and the patterns of keratin protein were analyzed by one- and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results indicated that the cultured airway cells synthesized additional keratins including 7, 15, 16, 17, and 18. However, consistent with the in vivo finding, fetal cells synthesized less or no keratin 13. These in vivo and in vitro studies strongly suggest that the synthesis of the keratin 13 in monkey conducting airway epithelium is developmentally regulated.     

人体上皮细胞系（株）间交叉污染的检测

本文根据不同上皮细胞的角蛋白丝性质和多肽组成的差异,建立了四种不同上皮细胞系(株)间交叉污染的检测方法:1.SDS-PAGE法;2.免疫印迹法;3.AE1单抗免疫荧光染色法;4.角蛋白丝结构转化法。结果表明:方法1—3比较有用。我们认为,要获得较满意的检测结果,需要根据不同上皮细胞的特点,选择不同的方法配合使用。     

Expression of hair-related keratins in a soft epithelium: subpopulations of human and mouse dorsal tongue keratinocytes express keratin markers for hair-, skin- and esophageal-types of differentiation

The dorsal surfaces of mammalian tongues are covered with numerous projections known as filiform papillae whose morphology varies in different species. Using a panel of monoclonal antibodies to keratins as probes, we have established that, in both human and mouse, the interpapillary epithelia express mainly the "esophageal-type" keratins, while the papillary epithelia express "skin-type" keratins as well as some keratins reacting with a monoclonal antibody (AE13) to hair keratins. The AE13-reactive proteins of the mouse were found to be very similar to those of authentic mouse hair keratins. However, the corresponding protein of human tongue appears to be different from all known human keratins. This protein has a MW of 51K; it is relatively acidic; it is sulfhydryl-rich, as revealed by iodoacetic acid-induced charge and apparent size shift; it shares an epitope with all the known acidic human hair keratins; and it is associated with keratin fibrils in vivo. This protein may therefore be regarded as a novel type I "hard" keratin. These data establish that mammalian dorsal tongue epithelia can be divided into at least three compartments that undergo mainly "esophageal-", "skin-" and "hair"-types of differentiation. Different keratin filaments, e.g., those of the esophageal- and hair-types, exhibit strikingly different degrees of lateral aggregation, which can potentially account for the different physical strength and rigidity of various cellular compartments. Our data also suggest the possibility that variations in papillary structure in human and mouse may arise from different spatial arrangements of specific keratinocytes, and/or from the expression of specialized hair-related keratins.     

Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris

From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I ("IE") keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 because its orthologs have been found in all jawed vertebrates (Gnathostomata) studied; in the tree, they form a common, most early branch, with the shark version, SstK18, in the most basal position. Thus, the sequences of SstK10 and SstK18 also favor the classical view of vertebrate evolution that considers the cartilaginous fishes as the most ancient living Gnathostomata. To determine the overall expression patterns of epidermal ("E") and simple epithelial ("S") keratins in this shark, we furthermore tested a panel of monoclonal anti-keratin antibodies by immunofluorescence microscopy of frozen tissue sections, and in immunoblots of cytoskeletal preparations, demonstrating that immunodetection of specific keratins is a convenient method to characterize epithelial tissues in shark.     

Characterization of bovine keratin genes: similarities of exon patterns in genes coding for different keratins.

Four different genomic clones which contain the genes coding for epidermal keratins Ia (mol. wt. approximately 68 000), Ib (68 000), III (60 000) and VIb (54 500) have been selected using cDNA probes and identified by hybrid-selection translation. The genes vary considerably in length, primarily due to differences in intron sizes: keratin Ia, 9.3 kb (approximately 2.55 kb total exons); keratin Ib, 6.0 kb (2.25 kb exons); keratin III, 6.0 kb (2.2 kb exons); keratin VIb, 4.4 kb (1.85 kb exons). The genes for all three representatives of the basic (type II) cytokeratin subfamily, i.e., keratins Ia, Ib and III, contain eight introns of variable sizes (0.1-1.8 kb) and their exon patterns are very similar. The gene coding for keratin VIb, a representative of the acidic (type I) subfamily, contains seven introns, and the size pattern of its five innermost exons closely resembles that of the genes of the type II keratins. Most of the introns are located in regions coding for the alpha-helical cores of these proteins. Mapping of the intron positions by the S1 nuclease technique and sequencing of some exon-intron boundaries has revealed that some of the introns of all four keratin genes have similar positions to each other and to those of the hamster vimentin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

An immunocytochemical study of keratin reactivity during rat odontogenesis.

The cytokeratin distribution in the developing rat enamel organ from day 15 of gestation through to 11 days post partum was examined immunohistochemically using a panel of monoclonal antibodies. A temporo-spatial programme of keratin expression was observed during odontogenesis and positive reactivity of the enamel organ was seen with the pan keratin antibodies CK1 (clone LP34 - reacts with a number of keratins including 6 and 18) and AE1-3 (reacts with most acidic and basic keratins). No reactivity was observed in the enamel organ with the other antibodies examined (Ks 8.12 [reacts with keratins 13 and 16], Ks 8.60 [reacts with keratins 10 and 11) and MCA157 [reacts with rat liver antigen]), although these antibodies did stain other epithelial tissues. This study supports the view that the epithelial cells of the enamel organ synthesize a tissue-specific subset of keratins which are related to the differentiation of the cells.