Keratin mRNA Research Articles

Generation and Characterization of Mirikizumab, a Humanized Monoclonal Antibody Targeting the p19 Subunit of IL-23.

Interleukin (IL)-23 exists as a heterodimer consisting of p19 and p40 and is a key cytokine for promoting inflammatory responses in a variety of target organs. IL-23 plays a key role in the differentiation and maintenance of T helper 17 cells, and deregulation of IL-23 can result in autoimmune pathologies of the skin, lungs, and gut. This study describes the generation and characterization of mirikizumab (miri), a humanized IgG4 monoclonal antibody directed against the p19 subunit of IL-23. Miri binds human and cynomolgus monkey IL-23 with high affinity and binds rabbit IL-23 weakly but does not bind to rodent IL-23 or the other IL-23 family members IL-12, IL-27, or IL-35. Miri effectively inhibits the interaction of IL-23 with its receptor, and potently blocks IL-23-induced IL-17 production in cell-based assays while preserving the function of IL-12. In both local and systemic in vivo mouse models, miri blocked IL-23-induced keratin mRNA or IL-17 production, respectively. These data provide a comprehensive preclinical characterization of miri, for which efficacy and safety have been demonstrated in human clinical trials for psoriasis, ulcerative colitis, and Crohn's disease. SIGNIFICANCE STATEMENT: This article describes the generation and characterization of mirikizumab, a high affinity, neutralizing IgG4 variant monoclonal antibody that is under development for the treatment of ulcerative colitis and Crohn's disease. Neutralization of interleukin (IL)-23 is achieved by preventing the binding of IL-23 p19 subunit to the IL-23 receptor and does not affect the IL-12 pathway.

In Vitro Induction of Epithelialization of Human Gingival Fibroblasts by Mesenchymal Stem Cells Conditioned Medium (MSCCM) and Its Effects on the Secretion of Interleukin-6 and Interleukin-1beta

Epithelial injury is difficult to restore normal structure and function upon self-repair. Tissue engineering technology provides hope for epithelial injury. It is showed that human gingival fibroblasts (HGFs) could be ideal seed cells for tissue repair. Our study intends to assess mesenchymal stem cell (MSC) conditioned medium’s effect on HGFs epithelization. HGFs were cultured In Vitro and divided into two groups, including control maintained at complete DMEM medium containing 10% FBS and induction group treated by MSC conditioned medium followed by analysis of cell proliferation by MTT assay, cell apoptosis, Keratin and vimentin expressions by real-time PCR and Western blot. IL-6 and IL-1β secretions by ELISA. MSC conditioned medium induction significantly inhibited HGFs proliferation, increased keratin mRNA and protein levels, reduced vimentin expression, and enhanced IL-6 and IL-1β secretions compared with control (P < 0.05). Caspase 3 activity failed to exhibit statistical significance (P > 0.05). MSC conditioned medium induced HGFs epithelization and promoted IL-6 and IL-1β secretions, thus providing theoretical basis for the repair function of HGFs.

Skin Stem Cells in Silence, Action, and Cancer.

In studying how stem cells make and maintain tissues, nearly every chapter of a cell biology textbook takes on special interest. The field even allows us to venture where no chapters have yet been written. In studying this basic problem, we are continually bombarded by nature's surprises and challenges. Stem cell biology has captured my interest for nearly my entire scientific career. Below, I focus on my laboratory's contributions to this fascinating field, to which so many friends and colleagues have made seminal discoveries equally deserving of this award.

Keratins Are Altered in Intestinal Disease-Related Stress Responses.

Keratin (K) intermediate filaments can be divided into type I/type II proteins, which form obligate heteropolymers. Epithelial cells express type I-type II keratin pairs, and K7, K8 (type II) and K18, K19 and K20 (type I) are the primary keratins found in the single-layered intestinal epithelium. Keratins are upregulated during stress in liver, pancreas, lung, kidney and skin, however, little is known about their dynamics in the intestinal stress response. Here, keratin mRNA, protein and phosphorylation levels were studied in response to murine colonic stresses modeling human conditions, and in colorectal cancer HT29 cells. Dextran sulphate sodium (DSS)-colitis was used as a model for intestinal inflammatory stress, which elicited a strong upregulation and widened crypt distribution of K7 and K20. K8 levels were slightly downregulated in acute DSS, while stress-responsive K8 serine-74 phosphorylation (K8 pS74) was increased. By eliminating colonic microflora using antibiotics, K8 pS74 in proliferating cells was significantly increased, together with an upregulation of K8 and K19. In the aging mouse colon, most colonic keratins were upregulated. In vitro, K8, K19 and K8 pS74 levels were increased in response to lipopolysaccharide (LPS)-induced inflammation in HT29 cells. In conclusion, intestinal keratins are differentially and dynamically upregulated and post-translationally modified during stress and recovery.

Arsenite suppression of BMP signaling in human keratinocytes

Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.

Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at day 15 of pregnancy.

Basal keratin expression in breast cancer by quantification of mRNA and by immunohistochemistry

Definitions of basal-like breast cancer phenotype vary, and microarray-based expression profiling analysis remains the gold standard for the identification of these tumors. Immunohistochemical identification of basal-like carcinomas is hindered with a fact, that on microarray level not all of them express basal-type cytokeratin 5/6, 14 and 17. We compared expression of cytokeratin 5, 14 and 17 in 115 patients with operable breast cancer estimated by real-time RT-PCR and immunohistochemistry.Despite the method of dichotomization and statistical analysis, there were cases with discordant results comparing immunohistochemistry and RT-PCR analysis. For dichotomisation based on quartiles and ROC, 14% of cases were negative on immunohistochemical examination for CK5/6, but presented high CK5 mRNA levels. There were also 48-55% cases, which were CK5/6-immunopositive, but were negative by mRNA examination. Similar discordances were observed for CK14 and CK17.Basal keratin mRNAs did not correlate with ER mRNA levels, while immunohistochemistry produced significant relationship with ER status.Our observation suggest that both method may produce different results in a small proportion of cases. Discordance between immunohistochemistry and RT-PCR may confound attempts to establish a simple methods for identification of basal-like tumors.

Reg IV enhances peritoneal metastasis in gastric carcinomas

The role of Regenerating (Reg) IV on peritoneal metastasis was examined in gastric cancer using. Reg IV-transfected human gastric cancer cells (MKN28-R1, MKN28-R2, TMK1-R1), control transfectants (MKN28-R0, TMK1-R0), and REG4-knocked down MKN45 cells were examined in in vitro and in nude mice peritoneal metastasis models. Increase of expression and secretion of Reg IV, and levels of BCL-2, BCL-XL,survivin, phosphorylated AKT, and phosphorylated EGFR, and decrease of nitric oxide-induced apoptosis were found in Reg IV-transfectants, whereas those were abrogated in the knockdown cells. In mice models, increased number and size of peritoneal tumors and decreased apoptosis were found in Reg IV-transfectants, whereas those were abrogated by the knockdown cells. Mice survivals were worsened in Reg IV-transfectants-inoculated mice, but were improved in Reg IV-knockdown cell-inoculated mice. Levels of Reg IV protein in peritoneal lavage fluids increased in Reg IV-transfectants inoculated mice, but decreased in Reg IV-knockdown cell inoculated mice. In metastasized human gastric cancers, Reg IV positivity in peritoneum-metastasis cases was higher than those in negative cases. Reg IV was detected in peritoneal lavage fluids from human gastric cancer patients, in whose lavages keratin mRNA was detected by reverse transcriptase-polymerase chain reaction. Collectively, Reg IV might accelerate peritoneal metastasis in gastric cancer. Reg IV in lavage fluids might be a good marker for peritoneal metastasis.

Long-term culture of keratinocyte-like cells derived from mouse embryonic stem cells

A number of epithelial lineages have been derived from mouse embryonic stem cells during the past decades, but the long lasting culture has never been reported. In this paper, we report when mouse embryonic stem cells were dispersed into small clumps containing approximately 50 to 100 cells and grown on mitotically inactivated mouse embryonic fibroblast feeder layers for up to 10 d to form epithelial-like colonies. Through subsequent cultivation without mouse embryonic fibroblast feeder layers, a serially subcultured keratinocyte-like cell lineage was established under these conditions. Pan cytokeratin, cytokeratin 14, and cytokeratin 18 were observed in these proliferating cells using immunocytochemistry and flow cytometry. E-cadherin, Involucrin, and keratin mRNAs were determined by a semi-quantitative and a quantitative real time reverse transcription-polymerase chain reaction (RT-PCR). These results confirmed the establishment of a keratinocyte-like cell lineage derived from mouse embryonic stem cells. In this paper also, we describe a method by which mouse embryonic stem cells can be differentiated into cells with some characteristics of epidermal keratinocytes and kept these cells in long-term culture. Potential applications of this method are the in vitro differentiation of cells of interest from embryonic stem (ES) cells of mice during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments or better understanding the mechanisms for epithelial differentiation of embryonic stem cells.

SiRNA‐Mediated Selective Inhibition of Mutant Keratin mRNAs Responsible for the Skin Disorder Pachyonychia Congenita

RNA interference offers a novel approach for treating genetic disorders including the rare monogenic skin disorder pachyonychia congenita (PC). PC is caused by mutations in keratin 6a (K6a), K6b, K16, and K17 genes, including small deletions and single nucleotide changes. Transfection experiments of a fusion gene consisting of K6a and a yellow fluorescent reporter (YFP) resulted in normal keratin filament formation in transfected cells as assayed by fluorescence microscopy. Similar constructs containing a single nucleotide change (N171K) or a three-nucleotide deletion (N171del) showed keratin aggregate formation. Mutant-specific small inhibitory RNAs (siRNAs) effectively targeted these sites. These studies suggest that siRNAs can discriminate single nucleotide mutations and further suggest that "designer siRNAs" may allow effective treatment of a host of genetic disorders including PC.

Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.

Pharmacology of RALGA, a Mixture of Retinaldehyde and Glycolic Acid

Background: Retinoids and α-hydroxy acids (AHAs) are major compounds in topical therapy. They exert distinct but potentially complementary activities. However, their association is limited by their respective irritating potential. Recently, the first association between a retinoid and an AHA has been achieved; this formulation (RALGA) associates retinaldehyde (RAL) – a precursor of retinoic acid (RA) – and glycolic acid (GA) – an AHA. Objective: To study the pharmacological properties of RALGA. Methods: The bioavailability of RAL into the skin after topical RALGA was studied by HPLC, and its bioconversion to RA was analysed by measuring the enzyme activity of retinaldehyde dehydrogenase and the RA content in the epidermis and dermis. The retinoid activity of RALGA was studied on the modulation of Hhb4 keratin mRNA on the tail of C57BL/6 mice, and its comedolytic properties on the size and density of dermal cysts and the morphology of sebaceous glands in hairless mice. Results: Epidermal and dermal concentrations of RAL and RA were higher after RALGA treatment, as compared to both RAL 0.1% alone and RA 0.05% alone; this indicates that the presence of GA favours the bioavailability and biotransformation of RAL into RA. The retinoid activity of RALGA (suppression of Hhb4 mRNA keratin) was similar to that of RAL alone, indicating that the presence of GA does not interfere with specific retinoid activity; GA alone had no effect in this test, which confirms the specificity of Hhb4 mRNA keratin modulation for retinoid activity. The diameter and the density of dermal cysts as well as the size of sebaceous glands were significantly decreased by RALGA. Conclusion: These observations indicate that the addition of an AHA such as GA to a retinoid such as RAL results in a better bioavailability of the retinoid, thus a higher delivery of RA, which potentiates the biological activities of the retinoid. This combination allows a delivery of high amounts of RA in the skin while preventing the side-effects usually observed with high concentrations of topical RA.

Keratin mRNA for detecting micrometastasis in cervical lymph nodes of oral cancer

We studied three keratin (K) gene candidates, K13, K19, and K20 mRNAs, for detecting micrometastases in cervical lymph nodes (LNs) by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 166 histologically metastasis-negative nodes, 24 micrometastatic LNs (14.4%) were detected based on K13 gene expression. Keratin 19 mRNA is an inadequate marker for the genetic diagnosis due to not only illegitimate gene expression from lymphatic tissue but also gene expression from the ectopic salivary gland. Keratin 20 mRNA showed low sensitivity. It is suggested that K13 mRNA may be a promising tumor marker among these keratin genes for detecting the micrometastases in cervical LNs of oral cancer.

Biological Activities of Topical Retinaldehyde

Background: We had hypothesised that retinaldehyde (RAL) should be an interesting precursor for topical use. Aim: We review our observations about its biological activities. Methods: We performed pilot studies to explore its biological effects and tolerability in human skin and compared the effects of topical RAL to that of all-trans-retinoic acid (RA) in the mouse tail test. Results: The biological activities of RAL were found to be qualitatively identical to that of RA: (i) induction of cellular RA-binding protein type 2 mRNA and protein, (ii) increase in epidermal proliferation (increase in DNA synthesis, epidermal thickness, induction of 50-kD keratin mRNA and reduction in 70-kD keratin mRNA), and (iii) metaplastic effects (induction of orthokeratosis, reduction of 65-kD keratin mRNA, increase in filaggrin and loricrin mRNAs). When associated with RAL, citral (known for its capacity to inhibit the oxidation of retinol to RA in epidermis) counteracted the effects induced by RAL indicating that RAL exerts biological activities through transformation to RA. Hypothesising that keratinocytes would metabolize 9-cis-RAL to 9-cis-RA, we compared the biological effects induced by topical 9-cis-RAL and found that hyperplastic and metaplastic responses were lower than those induced by all-trans-RAL or all-trans-RA at similar concentrations. This suggests that 9-cis-RAL has no advantage over all-trans-RAL for specific delivery of natural retinoids into the skin. As in clinical studies conducted in human skin, we also found topical RAL less irritant than RA. Conclusion: These studies indicate that topical RAL has biological activity and is well tolerated.

Localization of keratin mRNA and collagen I mRNA in gastric cancer by in situ hybridization and hybridization electron microscopy.

Localization of keratin mRNA and collagen I mRNA in gastric cancer by in situ hybridization and hybridization electron microscopy

In vivo footprints are found in the Xenopus 63 kDa keratin gene promoter prior to the appearance of mRNA

Previous work on the promoter region of the 63 kDa keratin gene demonstrated that in vivo footprints did not change during the transition from low-level to high-level transcription. Reverse transcription polymerase chain reaction and in vivo footprinting were used to determine whether these DNA–protein interactions are present before transcription begins. The results presented indicate that during development, DNA–protein interactions are present in the promoter region of the 63 kDa keratin gene at stage 44, four days prior to the initial appearance of 63 kDa keratin mRNA, at stage 48. Thus, the occupancy of these sites at stage 44 is not sufficient for transcription, but may have a role in `poising' the keratin promoter for the initiation of epidermal-specific transcription. The results suggest that the developmental history of a gene may be important in regulating its temporal and spatial expression.

The microanatomy, cell replication, and keratin gene expression of hair follicles during a photoperiod-induced growth cycle in sheep.

Exposure of New Zealand Wiltshire sheep to long days, following 24 weeks of short days, caused a synchronised out-of-season wool follicle growth cycle. Skin biopsies were collected at intervals between 3 and 30 days and follicles were examined by light microscopy in both transverse and longitudinal section to describe the regressive (catagen), resting (telogen) and regenerative (proanagen) stages of the induced growth cycle. Follicles were generally in the growing phase (anagen) during short day treatment but by day 20 after exposure to long day photoperiod. 16% of follicles were in late catagen. By day 52, all follicles were in various stages of catagen, telogen and proanagen. The progression through the cycle occurred more slowly, but was morphologically similar to follicle growth cycles reported in rodents and goats, induced by plucking or melatonin, respectively. Follicles in early catagen were rarely observed, possibly reflecting the brevity of this phase of the cycle. Late catagen follicles were distinguished by the presence of a brush end and an inner root sheath, the latter disappearing as follicles entered telogen. Immunocytochemistry of proliferating cell nuclear antigen provided evidence that mitotic activity in the follicle bulb ceased completely during the brief telogen phase. The simultaneous absence of type I intermediate filament keratin mRNA indicated that keratinocyte differentiation had also been interrupted. Cell proliferation was re-established in early proanagen prior to observable changes in the follicle microanatomy. The relatively synchronised follicle growth cycle induced by photoperiod manipulation represents a potentially useful model for the study of changes in follicle ultrastructure and the endocrine and biochemical regulation of seasonal hair growth patterns.

Programming gene expression in developing epidermis.

As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs throughout embryogenesis. To monitor transcriptional regulation, we coupled this with beta-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification. These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.

Wavelength-Specific Upregulation of Keratin mRNA Expression in Response to Ultraviolet Radiation

Keratin intermediate filaments are heteropolymers of coexpressed type I and type II protein chains, whose expression is tightly linked to the differentiation status of the keratinocyte. Epidermal basal keratinocytes coexpress keratins K5 and K14, whereas suprabasal keratinocytes downregulate K5 and K14 and begin to coexpress keratins K1 and K10. Using both isotopic and non-isotopic in situ hybridization, we have investigated the changes in expression of the messenger RNA species encoding the K5/K14 and K1/K10 keratin pairs in response to ultraviolet radiation. Here we report that following irradiation, the mRNA species encoding both keratin pairs is upregulated in a wavelength-specific manner, and that the link between the pattern of keratin mRNA expression and the differentiation status of the keratinocyte is disrupted. Forty-eight hours following ultraviolet B exposure, the amount of detectable mRNA encoding all four keratins studied had increased. Following UVA irradiation, the K1 and K10 signal increased to a much lesser extent than following ultraviolet B, whereas no change in the amount of mRNA encoding the K5/K14 pair was observed. Only two samples were examined following ultraviolet C exposure, but in both, increased K5/K14 signal, but not suprabasal K1/K10 signal, was observed. We suggest that the observations reported here may reflect important qualitative changes involved in photoadaptation of the epidermis, and provide further molecular markers of the different biological effects of ultraviolet radiation of different wavelengths.

Dietary Cysteine Regulates the Levels of mRNAs Encoding a Family of Cysteine-Rich Proteins of Wool

The abomasal or intravenous infusion of sulphur-containing amino acids such as cysteine or methionine into sheep on low-quality diets increases the sulphur content of the wool by increasing the synthesis of proteins containing a cysteine content of approximately 30 mol %. To investigate the molecular and cellular basis of this nutritional effect, quantitative analyses of wool keratin mRNA and protein levels, and follicle cortical cell type, were undertaken in sheep intravenously infused with cysteine. Northern blot analyses revealed that the mRNA levels of one gene family encoding cysteine-rich keratin-associated proteins (KAP4 family) expressed in the wool follicle cortex, increased approximately 5-6 times. Furthermore, the response was rapid as the mRNA levels increased approximately 3.5 times after 1 d of the cysteine infusion and, by 1 d post-infusion, they had fallen, approaching their basal level. No changes in the mRNA levels encoding the intermediate filament or the other keratin-associated protein families of lower cysteine content were observed. Concomitantly, two-dimensional polyacrylamide gel electrophoresis analysis of wool proteins showed a striking increase in the abundance of a group of cysteine-rich keratin-associated proteins in the wool by the end of the infusion period, returning to basal levels by 3 weeks later. At the cellular level, KAP4 expression was localized to the follicle paracortical cells, and the proportion of paracortical cells and the extent of KAP4 expression paralleled the changes in the cysteine infusion status of the sheep.