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E Fuchs

Publications and source records attributed to E Fuchs.

At least 55 records · Page 3Linked to original sources
PubMed1994

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.

Adaptor Protein Complex 2↗
PubMed1994

Mutations in the non-helical linker segment L1-2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex.

Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10 nm filaments, have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases, epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH). These diseases involve point mutations in either the basal epidermal keratin pair, K5 and K14 (EBS), or the suprabasal pair, K1 and K10 (EH). In severe cases of EBS and EH, mutations are found in the highly conserved ends of the alpha-helical rod domain, regions that, by random mutagenesis, had already been found to be important for 10 nm filament assembly. In order to identify regions of the keratin polypeptides that might be more subtly involved in 10 nm filament assembly and to explore the diversity in mutations within milder cases of these diseases, we have focused on Weber-Cockayne EBS, where mild blistering occurs primarily on the hands and feet in response to mechanical stress. In this report, we show that affected members of two different W-C EBS families have point mutations within 1 residue of each other in the non-helical linker segment of the K5 polypeptide. Genetic linkage analyses, the absence of this mutation in > 150 wild-type alleles and filament assembly studies suggest that these mutations are responsible for the W-C EBS phenotype. These findings provide the best evidence to date that the non-helical linker region in the middle of the keratin polypeptides plays a subtle but significant role in intermediate filament structure and/or intermediate filament cytoskeletal architecture.

Alleles↗
PubMed1994

Skin cancer and transgenic mice.

At the outer surface of the body, the epidermis of skin forms a protective barrier to keep microorganisms out and essential body fluids in. This barrier is composed of one main cell type, the keratinocyte, which undergoes a precisely defined program of differentiation to produce a durable and renewable integument. The delicate balance between differentiation and proliferation is maintained by regulatory molecules and growth factors. In basal and squamous cell carcinomas of the skin, the balance between growth and differentiation goes awry, although the detailed pathways underlying these cancers remain to be elucidated. Transgenic mice provide the opportunity to precisely perturb growth factors in the skin in the context of a whole organism, leading to valuable insight into both appropriate and deranged growth. This article will review the biology and biochemistry of epidermal growth and differentiation, with emphasis on basal and squamous cell carcinomas and hyperproliferative diseases of the skin, and how recent transgenic research has been utilized to take a penetrating look into these issues.

Animals↗
PubMed1993

An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.

Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.

Bacteriophage T7↗
PubMed1993

Postnatal development of central nervous alpha 2-adrenergic binding sites: an in vitro autoradiography study in the tree shrew.

The postnatal development of the alpha 2-adrenoceptor pattern was investigated by in vitro receptor autoradiography with the antagonist [3H]rauwolscine in the brains of tree shrews (Tupaia belangeri). At birth, high numbers of [3H]rauwolscine binding sites are diffusely distributed in the whole brain with exception of the neocortex which is very weakly labeled at this time. While the number of [3H]rauwolscine binding sites in the cerebellum decreases to low levels during the first three postnatal weeks, several brain regions show a significant increase in binding sites which are progressively concentrated in distinct nuclei. In the medulla oblongata, the diffuse labeling pattern changes so that binding sites become centralized in the dorsomedial nuclei. In the pons, similar changes can be observed with a moderate labeling of the locus coeruleus on postnatal day 10 and a strong labeling in the adult. In the thalamus, a transient appearance of high numbers of [3H]rauwolscine binding sites can be observed during the second and third postnatal week in specific nuclei. In the preoptic area and hypothalamus, there are only minor postnatal changes but the numbers of [3H]rauwolscine binding sites decrease between postnatal day 5 and adulthood. The high number of binding sites in the limbic system does not significantly change after birth. In the neocortex and the superior colliculus, the [3H]rauwolscine labeling pattern which is characteristic for the adult is achieved not before the third postnatal week. Competition experiments demonstrate that [3H]RAUW binds with high affinity to alpha 2-adrenoceptors in the postnatal as well as in the adult brain. Therefore, this study demonstrates region specific developmental profiles of the pattern of alpha 2-adrenoceptors in the postnatal tree shrew brain.

Aging↗
PubMed1993

The genetic basis of Weber-Cockayne epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical-stress-induced degeneration of basal epidermal cells. Recently, it was discovered that the more severe types, Dowling-Meara and Koebner, are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here, we show that the mildest type of EBS, Weber-Cockayne, is also a disorder of these keratins. Affected members of two unrelated families with Weber-Cockayne EBS had a T-->G point mutation in the second base position of codon 161 of one of two K5 alleles, leading to an Ile-->Ser mutation. This mutation was not present in unaffected members or in 156 alleles from normal individuals. Linkage analyses mapped the defect to the type II keratin gene cluster on chromosome 12q11-q13 (peak logarithm of odds score at theta = 0 of 3.0), providing strong additional evidence that this mutation is responsible for the Weber-Cockayne EBS phenotype. Conserved among type II keratins, Ile-161 is in the nonhelical head domain of K5, a region previously shown to be important for 10-nm filament assembly. The mutation generates a potential substrate site for protein kinase C, which could influence intermediate filament architecture, perhaps leading to the intrafilament association seen ultrastructurally in patients with the mutation.

Amino Acid Sequence↗
PubMed1993

Mefloquine compared with other malaria chemoprophylactic regimens in tourists visiting east Africa.

There is much confusion over which malaria chemoprophylaxis should be used in areas such as East Africa. We did two consecutive studies between 1985 and 1991 to assess the efficacy and side-effects of malaria chemoprophylaxis in short-term travellers to East Africa. All passengers returning from Kenya to Europe received an in-flight questionnaire and a second one three months later. Any report of documented malaria or of admission to hospital for possible side-effects was verified with the physician. 145 003 travellers completed questionnaires. Among the 139 164 who stayed in East Africa for less than one year, 296 cases of confirmed malaria were reported (275 due to P falciparum). In people who used no chemoprophylaxis, the incidence of falciparum malaria was 1.2% per month. Prophylactic effectiveness was 91% (95% Cl 85 to 94) for mefloquine, 82% (71 to 89) for pyrimethamine and sulfadoxine, 72% (56 to 82) for chloroquine plus proguanil, and 10 to 42% for chloroquine at various doses. Rates of side-effects, which were usually mild, were 18.8% for mefloquine users, 17.1% and 18.6% for chloroquine 300 mg and 600 mg base per week, respectively, 30.1% for chloroquine plus proguanil, and 11.7% for sulfadoxine and pyrimethamine. Mefloquine is significantly more effective than chloroquine plus proguanil for malaria prophylaxis in short-term tourists visiting East Africa and has a tolerance similar to that of chloroquine used alone.

Adult↗
PubMed1993

Disease severity correlates with position of keratin point mutations in patients with epidermolysis bullosa simplex.

Keratins are the major structural proteins of the epidermis. Recently, it was discovered that point mutations in the epidermal keratins can lead to the blistering skin diseases epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH), involving epidermal cell fragility and rupture upon mechanical stress. In this study, we demonstrate a correlation between disease severity, location of point mutations within the keratin polypeptides, and degree to which these mutations perturb keratin filament structure. Interestingly, of the 11 EBS or EH mutations thus far identified, 6 affect a single highly evolutionarily conserved arginine residue, which, when mutated, markedly perturbs keratin filament structure and keratin network formation. This site also appears to be a hot spot for mutation by CpG methylation and deamination. In the four epidermal keratins, there are several other CpG dinucleotides that exist at codons within the highly conserved ends of the keratin rod. To elucidate why mutations at these sites have not been detected in severe cases of EBS, we engineered 7 of these C-->T transitions in K14 and tested their ability to perturb keratin network formation and keratin filament assembly in vitro. The effects of these mutants on keratin filament network formation were significantly less severe than the EBS/EH arginine mutation, suggesting that the high incidence of mutations of the residue in EBS and EH patients is a result of both a special sensitivity of filament structure to perturbations in this residue and its susceptibility to mutagenesis.

Amino Acid Sequence↗
PubMed1993

Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. Synthesized by cells of the dermal component of skin, KGF's potent mitogenic activity is on the epidermal component, which harbors the receptors for this factor. To explore the possible role of KGF in mesenchymal-epithelial interactions in skin, we used a human keratin 14 promoter to target expression of human KGF cDNA to the stratified squamous epithelia of transgenic mice. Mice expressing KGF in their epidermis typically appeared frail and weak, and often had grossly wrinkled skin. These mice exhibited a gross increase in epidermal thickness accompanied by alterations in epidermal growth and differentiation. Most remarkably, animals displayed several striking and unexpected changes, including a marked suppression of hair follicle morphogenesis and suppression of adipogenesis. With age, some animals developed gross transformations in the tongue epithelium and in epidermis. In addition, they exhibited elevated salivation and their salivary glands showed signs of altered differentiation. Collectively, our findings provide new and important insights into the roles of KGF, implicating this potent growth factor in eliciting global effects not only on growth, but also on development and differentiation, of skin and other tissues. In particular, KGF seems to interfere with signalling of some mesenchymal-epithelial interactions.

Animals↗
PubMed1993

Prevention of runting and cachexia by a chimeric TNF receptor-Fc protein.

Tumor necrosis factor alpha (TNF) is an important mediator of septic shock and cachexia. A soluble form of the human type 2 TNF receptor, constructed by joining the Fc region of human IgG1 to the TNF receptor, prevents weight loss in nude mice bearing a TNF-secreting tumor. This soluble receptor was also used to treat TNF transgenic mice which were runting and died before reaching reproductive age. After continuous treatment with soluble TNF receptor, the TNF transgenic mice grew to normal size and reproduced. Thus, soluble TNF may be useful in counteracting the detrimental systemic effects of TNF in a clinical setting.

Animals↗
PubMed1993

Cyclosporine-induced syngeneic graft-vs-host disease: prevention of autoaggression by treatment with monoclonal antibodies to T lymphocyte cell surface determinants and to MHC class II antigens.

Administration of cyclosporine (CsA) following syngeneic/autologous bone marrow transplantation (BMT) elicits a T lymphocyte-dependent autoimmune disease resembling graft-vs-host disease (syngeneic GVHD). This autoaggression syndrome appears to be due to the autorecognition of self-MHC class II antigens by CD8+ cytolytic T cells and a CD4+ autoreactive T cell subset. The syngeneic GVHD model was used to assess the effectiveness of treatment with monoclonal antibodies to the alpha/beta T cell receptor (TCR) and the CD4 or CD8 determinants on the prevention of autoimmune disease. Nylon wool nonadherent splenic T cells (50 x 10(6)) from Lewis strain rats with active syngeneic GVHD were adoptively transferred into irradiated (1050 rad syngeneic recipients reconstituted with normal marrow (60 x 10(6) cells). Monoclonal antibody (McAb) to the alpha/beta TCR, the CD4 determinant, or the CD8 determinant was administered to secondary recipients on Days 0, 3, 6, 9, and 12 at a dose of 0.1 ml of ascites fluid. Control animals received normal mouse serum on the same schedule. Animals treated with either saline or normal mouse serum developed syngeneic GVHD within 16-20 days. Comparatively, syngeneic GVHD developed much later in the secondary recipients treated with anti-CD4 McAb (onset of syngeneic GVHD, 28-32 days) and was less severe compared to the control group. On the other hand, the recipients treated with McAb's to the alpha/beta TCR or to the CD8 determinant did not develop syngeneic GVHD (monitored over 10 weeks post-therapy). Peripheral blood lymphocytes from these recipients also were analyzed for T cell subsets by phenotypic analysis. There was a pronounced reduction of the total number of cells expressing the alpha/beta TCR and the CD8 determinant after treatment of the recipients with the McAb's to the alpha/beta TCR and to the CD8 determinant, respectively. Recovery to normal levels began to occur 6 weeks after the last dose of McAb. There was a significant reduction of the CD4+ subset after treatment with anti-CD4 McAb, but it was not long lasting with recovery coinciding with the onset of syngeneic GVHD. Studies were also performed to evaluate McAb therapy of established syngeneic GVHD. The McAb's were found to be largely ineffective due in part to pulmonary toxicity. Furthermore, this model was utilized to evaluate the efficacy of treatment with McAb to the target antigen of syngeneic GVHD. Infusion of McAb to a public determinant on class II MHC molecules prevented or significantly delayed the onset of syngeneic GVHD after adoptive transfer of effector cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Postnatal development of 3H-rauwolscine binding sites in the dorsal lateral geniculate nucleus and the striate cortex of the tree shrew (Tupaia belangeri).

Noradrenaline has been shown to play an important role within the visual system of the brain. To analyze the postnatal development of alpha2-noradrenergic receptors in the visual system of tree shrews, we localized and quantified binding sites for the antagonist [3H]-rauwolscine by in vitro-autoradiography in the dorsal lateral geniculate nucleus and the striate cortex at different postnatal ages. At birth, the dorsal lateral geniculate nucleus is only slightly labeled by [3H]-rauwolscine. During the postnatal period, the number of binding sites increases to reach a maximum around postnatal day 20. Since the young tree shrews open their eyes at approximately day 19, it appears that this high concentration of alpha2-adrenoceptors is related to eye opening. In the adult animal, [3H]-rauwolscine labeling shows a laminated pattern in the dorsal lateral geniculate nucleus. Laminae 1, 2, and 3 are more strongly labeled than laminae 4, 5, and 6. In the striate cortex, the pattern of [3H]-rauwolscine-binding sites changes dramatically during the early postnatal period. Immediately after birth, there is only one layer, located within the subplate zone, which is labeled. From postnatal day 5 onwards, all cortical layers which can be distinguished on histologically stained sections reveal [3H]-rauwolscine-binding sites, but in layer IV, which is known to receive major inputs from the dorsal lateral geniculate nucleus, there is very little labeling during the first two postnatal weeks. In this layer, a large number of [3H]-rauwolscine-binding sites occurs between postnatal day 15 and 20, that is slightly before and around the time of eye opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Autoradiographic localization of prazosin and rauwolscine binding sites in the human kidney.

In the human kidney, binding sites for the alpha 1-adrenoceptor antagonist 3H-prazosin and the alpha 2-adrenoceptor antagonist 3H-rauwolscine were localized and quantified by in vitro autoradiography. 3H-prazosin binding was found predominantly in the renal cortex. In the medulla, tubular structures were also specifically labelled. No binding sites, however, were detected in association with glomeruli or large blood vessels. 3H-rauwolscine labelled the medullary vascular bundles intensively, but no binding sites were associated with glomeruli or other cortical structures. Thus, the binding pattern for 3H-prazosin is quite similar in both human and rat renal cortex. There are, however, distinct differences between human and rat kidneys in the distribution of the alpha 2-adrenergic binding sites visualized with the antagonist 3H-rauwolscine.

Animals↗
PubMed1993

Psychosocial conflict in the tree shrew: effects on sympathoadrenal activity and blood pressure.

We investigated the influence of psychosocial conflict (PSC) on systolic blood pressure (BP) and on sympathoadrenal activity in male tree shrews. BP was recorded and urinary epinephrine and norepinephrine were determined daily during a 10-day control period and a subsequent 10-day period of PSC. At the end of the experiments, levels of adrenal tyrosine hydroxylase (TH), phenylethanolamine-N-methyl transferase (PNMT), epinephrine, and norepinephrine were determined. The reactivity of the sympathoadrenal system and of BP depended on the social position achieved. In subordinates, urinary norepinephrine excretion was constantly elevated while epinephrine excretion was elevated only transiently. Adrenal norepinephrine, epinephrine, and TH were increased whereas PNMT remained unaffected. Despite the sympathoadrenal arousal, the increase in BP was only temporary. In dominant animals, PSC had no effects on BP, adrenal parameters or urinary norepinephrine. These results reinforce the concept of distinctive neuroendocrine and hemodynamic response patterns to psychosocial stimuli depending on the type and degree of control which an individual can exert over the challenge.

Adrenal Glands↗
PubMed1993

Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association.

Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.

Amino Acid Sequence↗
PubMed1993

The roles of the rod end and the tail in vimentin IF assembly and IF network formation.

Using mutagenesis, we investigated the importance of two vimentin domains: (a) a highly conserved segment near the carboxy end of the alpha-helical rod, and (b) the tail, with which the rod end is known to interact. As judged by in vitro filament assembly and expression in transiently transfected cells lacking an endogenous vimentin network, the rod-tail interaction is not essential for 10 nm filament structure in vitro or for formation of fibrous arrays in culture. However, when mutated, amino acid residues within the rod and the tail segments can cause perturbations in IF assembly and in IF network formation. Finally, our studies show that the vimentin tail seems to play a role both in thermodynamically stabilizing IF structure in vitro and in establishing proper IF networks in vivo.

Amino Acid Sequence↗
PubMed1993

Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice.

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

3T3 Cells↗