Two signal model of lymphocyte activation.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
E Fuchs
Publications and source records attributed to E Fuchs.
Distribution of 125I-endothelin-1,-2,-3 binding sites in mammalian kidneys.
1. Binding sites for 125I-labeled endothelin (ET) isopeptides ET-1, ET-2 and ET-3 were visualized by autoradiography in the kidneys of man, baboon, rhesus monkey, tree shrew, pig and rat. 2. Highest levels of binding for the three isoforms were observed in the glomeruli, cortical and medullary vessels of baboon, rhesus monkey, pig and rat, whereas there was no noticeable labeling of glomeruli in human and tree shrew kidneys. 3. The enrichment of binding sites depended on the species and peptide investigated, suggesting different affinities and/or densities of a heterogenous population of renal ET receptors.
Animals↗
The molecular biology of intermediate filament proteins.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations.
Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.
Amino Acid Sequence↗
The roles of K5 and K14 head, tail, and R/K L L E G E domains in keratin filament assembly in vitro.
Type I and type II keratins form obligatory heterodimers, which self-assemble into 10-nm intermediate filaments (IFs). Like all IF proteins, they have a central alpha-helical rod domain, flanked by nonhelical head and tail domains. The IF rod is more highly conserved than head and tail, and within the rod, the carboxy R/K L L E G E sequence is more highly conserved than most other regions. Mutagenesis studies have shed some light on the roles of the head, tail, and R/K L L E G E sequence in 10-nm filament structure. However, interpretations have often been complicated in part because many of these studies have focused on transfected cells, where filament structure cannot be evaluated. Of the few in vitro assembly studies thus far conducted, comparison of keratin mutants with other IF mutants have often been difficult, due to the obligatory heteropolymeric nature of keratin IFs. In this report, we describe in vitro filament assembly studies on headless, tailless, headless/tailless, and R/K L L E G E truncated mutants of keratin 5 and its partner keratin 14. Using varying conditions of ionic strength and pH, we examine effects of analogous K5 and K14 mutations on the stability of 10-nm filament structure. Using EM, we examine effects of mutations on the ability of subunits/protofibrils to (a) elongate and (b) laterally associate. Our results demonstrate that (a) tails of K5 and K14 are required for filament stabilization; (b) the head of K5, but not of K14, is required for filament elongation and lateral alignments; and (c) the R/K L L E G E domains are required for lateral alignments, but not for filament elongation.
Amino Acid Sequence↗
Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice.
Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.
Animals↗
Genetic skin disorders of keratin.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.
The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.
Animals↗
Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.
Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.
Base Sequence↗
Autoradiographic characterization of angiotensin receptor subtypes in fetal and adult human kidney.
To better understand the action of angiotensin II (ANG II) and angiotensin receptor antagonists (ARA) in human kidney, ANG II receptors were characterized by in vitro autoradiography in fetal and adult human renal tissue using 125I-[Sar1-Ile8]ANG II (125I-ANG II), a potent ANG II antagonist. Binding was inhibited with the ARAs DuP 753 and PD 123177, respectively. In adult kidneys (n = 5), binding of 125I-ANG II showed the following characteristics: arterial vessels had dissociation constant (Kd) = 387.6 +/- 29.1 (SD) pM and maximal binding (Bmax) = 41.4 +/- 3.6 fmol/mg tissue equivalent (TE); glomeruli had Kd = 885.7 +/- 217.1 pM and Bmax = 35.5 +/- 8.1 fmol/mg TE; and outer medulla had Kd = 142.1 +/- 52.5 pM and Bmax = 7.7 +/- 2.3 fmol/mg TE. PD 123177 effectively displaced 125I-ANG II only in large preglomerular vessels [half-maximal inhibitory concentration (IC50) = 0.22 +/- 0.1 nM, type 2 receptor (AT2)], whereas DuP 753 displaced only the labeled ligand in glomeruli (IC50 = 0.28 +/- 0.11 nM) and outer medulla (IC50 = 0.39 +/- 0.11 nM, AT1). In fetal kidneys (n = 4), a diffuse 125I-ANG II binding was demonstrated in the medulla (Kd = 36.6 +/- 7.1 pM; Bmax = 25 +/- 3.8 fmol/mg TE) and in the cortex (Kd = 19.5 +/- 8 pM; Bmax = 7.2 +/- 2.2 fmol/mg TE). Both cortical (IC50 = 0.039 +/- 0.019 nM) and medullary binding (IC50 = 0.076 +/- 0.039 nM) could only be displaced by PD 123177.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Screening of blood donors and recipients for Borrelia burgdorferi antibodies: no evidence of B. burgdorferi infection transmitted by transfusion.
In the study presented here, the prevalence of antibodies against Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was determined in a group of blood donors from the Würzburg area (Southern Germany). 13 of 472 donors (2.7%) tested were positive by immunoblotting (IB). These 13 donors were examined in more detail by physical examination, anamnesis and determination of inflammation parameters of the blood. All persons were asymptomatic for Lyme borreliosis. One of 5 who remembered a tick bite actually had suffered from an erythema chronicum migrans 5 years ago. Another one had been affected by fever, headaches and pain in the limbs, arthralgia and motoric disorder in both hands 6 months before examination. Analysis of the blood did not provide any evidence of an acute infection. Moreover, each of the 472 serum samples was analyzed by a hemagglutination test (HAT). 26 (5.5%) showed a positive test result. In order to investigate whether a seroconversion of the recipients by transfusion of B. burgdorferi antibody-positive blood had taken place, 9 recipients of blood products originating from the 13 IB-positive donors were serologically reexamined. All samples taken proved to be antibody-negative. Consequently, the transfusion did not produce any seroconversion in the patients thus treated.
Antibodies, Bacterial↗
[Infections with Borrelia burgdorferi in Würzburg blood donors: antibody prevalence, clinical aspects and pathogen detection in antibody positive donors].
The prevalence of antibodies against Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was determined in a group of blood donors from the Würzburg area (Southern Germany). 26 of 472 donors (5.5%) tested positive in a hemagglutination test. When performing immunoblots only 13 donors (2.7%) gave rise to B. burgdorferi-specific antibodies. 9 of them were examined in more detail by anamnesis, physical examination, determination of inflammation parameters of the blood and polymerase chain reaction (PCR) analysis of urine. All persons were asymptomatic for Lyme borreliosis. One of 4, who remembered a tick bite, actually had suffered from an erythema migrans 5 years ago. Another one had been affected by fever, headaches and pains in the limbs, arthralgia and motoric disorder in both hands 6 months before examination. Analysis of the blood did not provide any evidence of an acute infection. In the urine of 2 donors we detected B. burgdorferi-specific DNA by PCR. No seroconversion due to blood transfusion could be observed, when 9 recipients of blood products provided by the 13 seropositive donors were serologically reexamined. PCR analysis of urine samples of 5 recipients was also negative.
Antibodies, Bacterial↗
Quantification and excretion profiles of pteridines in primate urine.
Biopterin, 6-hydroxymethyl-pterin, isoxanthopterin, neopterin and, pterin were quantified in stress-free collected spontaneous morning urine samples from Callithrix jacchus, Saguinus fuscicollis, Saguinus labiatus, Saimiri sciureus, Presbytis entellus, Cercopithecus albogularis, Cercocebus torquatus, Macaca fascicularis, Hylobates concolor, Pongo pygmaeus, and Gorilla gorilla. In most species, biopterin was the most frequent urinary pteridine followed by neopterin. Sex differences in biopterin and neopterin excretion were observed in Gorilla gorilla and Pongo pygmaeus. Pterin and isoxanthopterin were only present in minor concentrations. 6-hydroxymethyl-pterin was barely detectable and not present in the urine of Saguinus labiatus, Saimiri sciureus, and both male Gorilla gorilla and Pongo pygmaeus.
Animals↗
Localization and characterization of IGF-I receptors in fetal and adult human kidneys.
Human insulin-like growth factor I (IGF-I) is a growth and differentiating factor produced by various adult and fetal tissues. In the kidney, it has been linked to the proliferative response of renal tubular and glomerular cells, following unilateral or partial nephrectomy, in acromegaly, in diabetes mellitus and in glomerulonephritis. To gain insight into the potential effects of IGF-I in human kidney, a quantitative analysis of IGF-I-binding sites was performed in fetal and adult tissue using 125I-IGF-I. The ligand consistently labelled renal cortex, medulla, and glomeruli while renal vessels were not uniformly marked. The highest affinity of binding sites was found in glomeruli (adult kidneys: Kd 24.7 +/- 5.1 pM; Bmax 5.2 +/- 0.5 fmol/mg tissue equivalent (TE); n = 4; fetal kidneys: Kd 17.0 +/- 2.5 pM; Bmax 4.5 +/- 0.7 fmol/mg TE; n = 4) and cortical tubules, while vessels and renal medulla (adult kidneys: kd 47 +/- 3.9 pM, Bmax 2.6 +/- 0.3 fmol/mg TE; n = 4; fetal kidneys: kd 41.6 +/- 9.2 pM, Bmax 3.5 +/- 0.4 fmol/mg TE; n = 3) had only about half the affinity of binding and a significantly reduced maximal capacity. The strong binding of 125I-IGF-I to glomeruli supports the view that IGF-I may be involved in modulating glomerular structure and function. Fetal renal growth may depend on the action of IGF-I on glomerular cells and tubular epithelia of the kidney.
Autoradiography↗
The expectations of the medical profession concerning the EEC directives for allergenic extracts.
Explore the source record for details and available documents.
Allergens↗
Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.
Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.
Amino Acid Sequence↗
Transcription factor AP2 and its role in epidermal-specific gene expression.
The epidermis is a stratified squamous epithelium whose major differentiation-specific products are keratins. To elucidate factors controlling keratinocyte-specific gene expression, we previously identified proximal and distal regulatory elements that act synergistically to drive keratinocyte-specific expression of the gene encoding human epidermal keratin K14. Control by the proximal element is mediated by a transcription factor, KER1, which is more abundant in nuclear extracts of keratinocytes than in extracts of other cell types, including fibroblasts, lymphocytes, and simple epithelial cells. In this report, we identify this factor as transcription factor AP2, shown to be transcribed in cells of epidermal and neural crest lineages. Furthermore, we demonstrate functional AP2 binding sites upstream from three additional epidermal genes, suggesting that AP2 may be generally involved in epidermal gene regulation. Finally, although AP2 is necessary, it is not sufficient for epidermal gene expression: a distal element contributes to tissue-specific expression of the human keratin K14 gene as judged by its ability to enhance expression of a heterologous promoter in keratinocytes but not in hepatoma cells. These results imply that a combination of factors is likely to contribute to epidermal-specific expression of the human keratin K14 gene.
Base Sequence↗
Retinoid-mediated transcriptional regulation of keratin genes in human epidermal and squamous cell carcinoma cells.
Vitamin A and other retinoids profoundly inhibit morphological and biochemical features of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, we examined retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. Our studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5' upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors alpha and gamma, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.
Carcinoma, Squamous Cell↗