Keratin expression in mouse epidermal tumors.
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Animals↗
Biomedical subjects
C K Cheng
Publications and source records attributed to C K Cheng.
Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis.
Monospecific antikeratin antisera and specific complementary DNA probes were used to analyze expression of keratin genes in newborn mouse skin and skin papillomas and carcinomas by indirect immunofluorescence, immunoblotting, and in situ hybridization. Tumors were induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Type I epidermal keratin K14 protein (Mr 55,000) is found in all living layers of the newborn skin but is most abundant in the lower strata. K1 (Mr 67,000) and K10 (Mr 59,000) proteins are predominantly suprabasal and K1 is processed in the stratum corneum. Transcripts for K14 were confined largely to the basal cell layer by in situ hybridization. Transcripts for K1 and K10 were highly expressed in suprabasal cells including the granular cell layer. In benign tumors, distribution of K14 protein is similar to that in newborn skin, while the abundance of K1 and K10 appears to be somewhat reduced although the tissue distribution remains suprabasal. Transcription of K14 is aberrant in benign tumors and transcripts persist throughout much of the suprabasal cell layers. Transcripts of K1 and K10 are normally distributed in papillomas but grain density is less intense than in newborn epidermis. Keratin expression in carcinomas is highly disturbed. K14 protein and transcripts are highly expressed in all strata in carcinomas while protein and transcripts for K1 and K10 are essentially absent. These results suggest that papilloma cells fail to respond to or generate signals to regulate K14 expression in the differentiating suprabasal cell layers and may not fully express their suprabasal cell keratins. Carcinomas fail to express suprabasal cell keratins and this is regulated at the transcriptional level. The loss of suprabasal keratin expression may provide a marker for malignant conversion in the mouse skin carcinogenesis model.
9,10-Dimethyl-1,2-benzanthracene↗
Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis.
A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.
Amino Acid Sequence↗
An extrachromosomal plasmid is the etiological precursor of kalDNA insertion sequences in the mitochondrial chromosome of senescent neurospora.
In the kalilo strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nucleotide sequence, kalDNA, into mitochondrial DNA. A 9.0 kb linear DNA plasmid that is structurally homologous to the mitochondrial kalDNA insertion sequences exists in high copy numbers in close association with the nuclei of presenescent and senescent kalilo cells, but is not present in cells of long-lived normal strains. The free kalilo plasmid has not been detected in mitochondria, suggesting that the element does not contain a mitochondrial origin of replication. Unexpectedly, the nuclear plasmid, like the mitochondrial insertion element, follows a strict pattern of maternal inheritance. We surmise that the extramitochondrial plasmid is the etiological precursor of the kalDNA insertion sequences that appear in the mtDNAs of senescent cell lines and conclude that the kalilo element induces senescence because it is a mutator of mitochondrial genes.
DNA Replication↗
Regulated synthesis of low-molecular-weight antigens in keratinocyte cell cultures.
Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low Ca++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [35S]methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32PO4, the 11K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocytes suggests that these proteins are important in keratinocyte proliferation and differentiation.
Animals↗
Organization of a type I keratin gene. Evidence for evolution of intermediate filaments from a common ancestral gene.
The genomic structure of the mouse 59-kDa keratin gene, a Type I intermediate filament (IF) gene is presented. A comparison of the organization of this gene with that of the human 67-kDa keratin, a Type II IF gene, and hamster vimentin, a Type III IF gene, suggests a common evolutionary origin for Type I, II, and III IF genes. Most introns in these three types of IF genes occur at similar positions within the region encoding sequences predicted to form coiled-coils, but do not delineate structural subdomains. Interestingly though, most of the introns interrupt at or near the beginning of the characteristic 7-residue (heptad) repeat of sequences which form the coiled-coil. These data suggest that the three types of IF genes arose from a common ancestor which may have been assembled from smaller units containing multiple heptad repeats. Subsequent duplication events may then have formed the three known alpha-helical types and each of their various members.
Animals↗
The use of cDNA clones and monospecific antibodies as probes to monitor keratin gene expression.
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Amino Acid Sequence↗
Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies.
The presence of common antigenic determinants within all keratin proteins has made difficult the production of antisera which are monospecific for individual keratin subunits. Synthetic peptides corresponding to the carboxyl-terminal amino acid sequences of the mouse 59- and 67-kilodalton keratins were used to produce antibodies which were highly specific for these keratin subunits. This method of antibody production was chosen after examination of amino acid sequence data (which were deduced from the nucleotide sequence of cDNA clones for these and other mouse keratins) revealed that the carboxyl-terminal amino acid sequences of various keratins were unique. Indirect immunofluorescence staining of newborn-mouse skin with these antisera demonstrated that the 59- and 67-kilodalton keratins were only present within the differentiated cells of the epidermis (the suprabasal layers) and not in the undifferentiated cells (the basal layer). These results are consistent with our previous work concerning the expression of these keratin genes at the messenger RNA level (Roop, D. R., Hawley-Nelson, P., Cheng, C. K., and Yuspa, S. H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 716-720). Data obtained with the antisera directed against the 67-kilodalton keratin also indicated that the carboxyl-terminal sequences of this subunit were not present in the nonliving layer (the stratum corneum) of the epidermis. This approach should be useful for the production of antisera specific for other keratin subunits as additional sequence information becomes available.
Amino Acid Sequence↗
Keratin gene expression in mouse epidermis and cultured epidermal cells.
The major differentiation products of mouse epidermis are keratins of 40-70 kilodaltons (kDal). We have prepared a library of cDNA clones from total poly(A)+ RNA from newborn mouse epidermis. Clones corresponding to the major in vivo keratins of 55, 59, and 67 kDal have been isolated and characterized. By RNA blot analysis of poly(A)+ RNA from newborn mouse epidermis, we have identified RNA species that are approximately 1,600, 2,000, and 2,400 nucleotides in length and are complementary to the cDNAs for the 55-, 59-, and 67-kDal keratins, respectively. Analysis of RNA from primary cultures of newborn mouse epidermis by this same technique shows greatly reduced levels of these RNAs. Transcripts complementary to all three cloned cDNAs are abundant in 14- to 16-day embryonic and adult mouse skin. Thus, altered expression in culture does not appear to be due to induction of a developmentally programmed switch by placing the cells in culture but instead is due to factors modulating expression within the culture system. Because the 55-, 59-, and 67-kDal keratins are the major proteins in epidermis they probably represent keratin associated with terminal differentiation. The expression data suggest that cultured cells are blocked in expression of differentiation keratins but instead synthesize other keratin family members probably related to cytoskeletal functions.
Animals↗
Expression of keratin genes in mouse epidermis and normal and malignantly transformed epidermal cells in culture.
Complementary DNA (cDNA) clones constructed to the 55, 59 and 67 kilodalton (K) keratins, the major keratins synthesized in newborn mouse epidermis, were used as molecular hybridization probes to examine the expression of these genes in newborn epidermis and normal and malignantly transformed epidermal cells in culture. Transcripts of these three keratin genes are abundant in newborn epidermis. However, primary cultures of epidermal cells contain very low levels of these RNAs. The decreased expression of these keratin genes in primary cells appears to be due to factors within the culture system. Unlike primary-cell cultures, the malignantly transformed cell line Pam 212 synthesizes keratin proteins and mRNAs similar to newborn epidermis, including the 67 K keratin. However, synthesis of the 67 K keratin in Pam 212 cells is modulated by culture factors. Keratin gene expression in another Pam line, 321, differs from that of Pam 212 cells in that decreased expression of these three keratin genes occurs. These results indicate that keratin genes that are normally expressed in vivo in epidermis may be expressed in malignant epidermal cells under conditions that do not permit expression of these genes in nonmalignant primary epidermal cells.
Animals↗
Rocket immunoelectrophoresis in the presence of denaturing agents.
Modifications of the Laurel rocket technique for assaying antigen antibody reactions are described. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents (e.g., SDS and urea) and subjected to electroimmunoassay without loss of sensitivity or specificity. Methods are also presented for obtaining rockets using gel slices from polyacrylamide gel electrophoresis either in the presence of urea or SDS. Results obtained using keratins, the insoluble proteins of epidermis, hair and nail are summarized.
Animals↗
The keratin polypeptides of psoriatic epidermis.
The polyacrylamide SDS electrophoretic pattern of protein extracted from the stratum corneum obtained by scraping the surface of involved skin of patients with psoriasis was different from that of uninvolved skin and normal controls. The pattern from superficial scales was similar to that of whole stratum corneum in the case of involved psoriatic epidermis but different in uninvolved and normal epidermis. These data indicate that the changes which are observed in the structural proteins during normal keratinization are not seen in involved psoriatic epidermis. In addition, the relative proportion of keratin polypeptides was different in involved psoriatic epidermis compared to normal skin. That these changes are not specific for psoriasis was shown by finding similar electrophoretic patterns with stratum corneum proteins from patients with other keratinizing disorders.
Electrophoresis, Polyacrylamide Gel↗
Erucic acid metabolism by rat heart preparations.
Rat heart preparations metabolized erucic acid at much slower rates than palmitic acid. This applied for activation reaction, for the conversion of acyl-CoA to acylcarnitine, and for the utilization of acyl group for oxidation. As compared to palmityl-CoA, erucyl-CoA exhibited a lower affinity for carnitine palmityltransferase (EC 2.3.1.23), the respective apparent Michaelis constants were 43 and 83 muM. Presence of erucyl-CoA or erucyl-carnitine slowed the mitochondrial oxidation of palmityl groups apparently because of the slower oxidation of erucyl groups. However, presence of erucate did not inhibit the activation of palmitate. Heart mitochondria obtained from rats fed rapeseed oil (50 cal %) or corn oil diet for 3 days showed similar abilities for the coupled oxidation of various substrates and similar carnitine palmityltransferase activities. Thus, a suggestion of gross mitochondrial malfunction following rapeseed oil consumption was not confirmed.
Animals↗
Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum).
The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10(-6)-10(-4)m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10(-4)m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification.
Journal Article↗
The vectorcardiogram of healthy young Chinese. Frank lead system.
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Adult↗
Alzheimer's disease care: costs and potential savings.
A cross-sectional study of 679 Alzheimer's disease patients from thirteen sites in nine states provides a unique opportunity to estimate costs of Alzheimer's disease care by disease stage and care setting and to explore potential areas of cost savings. In 1996 annual costs of caring for patients with mild, moderate, and severe Alzheimer's disease were $18,408, $30,096, and $36,132, respectively. Monthly savings of $2,029 in formal services are possible if disease progression can be slowed. Annual institutional cost savings of $9,132 also are achievable if alternative residential settings are used.
Aged↗
A case of pancreatic serous cystadenoma obstructing the distal pancreatic duct.
We present a case of resected serous cystadenoma of the pancreas inducing marked dilatation of the main distal pancreatic duct. A 68-year-old woman, previously diagnosed with chronic pancreatitis, presented with upper abdominal pain. Abdominal US revealed a highly echoic mass in the pancreas. A CT scan disclosed a low density mass in the pancreas and dilatation of the main peripheral pancreatic duct. The mass demonstrated homogeneous and high signal intensity on T2-weighted magnetic resonance imaging (MRI). Selective abdominal arteriography showed the mass strained by the celiac artery. The tumor markers were CEA (2.4 ng/ml) and CA19-9 (6.1 U/ml). After the diagnosis of serous cystadenoma of the pancreas, the patient underwent distal pancreatectomy and splenectomy. The tumor (2.5 cm in diameter) consisted of grayish-white nodules and occupied the body of the pancreas. The tail of the pancreas was atrophic. Histopathological examination of the specimen showed a multilocular lesion containing numerous cysts with the inner surfaces evenly lined by one layer of cuboid or flat epithelial cells which stained positive for periodic acid-Schiff (PAS), evidencing serous cystadenoma. The patient is doing quite well one and a half years after the operation.
Aged↗