Biomedical subjects

E Fuchs

Publications and source records attributed to E Fuchs.

At least 37 records · Page 2Linked to original sources

PubMed1994

Hippocampal glucocorticoid receptor expression in the tree shrew: regulation by psychosocial conflict.

1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri). 2. To generate probes for the in situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence. 35S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus. 3. After in situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus. 4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.

Amino Acid Sequence↗

PubMed1994

The epidermis: rising to the surface.

At the skin surface, the epidermis serves an important protective function which it manifests by building an extensive cytoskeletal architecture of keratin filaments, spanning from the nuclear envelope to hemidesmosomes and desmosomes. Recent studies on epidermal proteins and their interactions have provided insights into human skin diseases, including genetic disorders of keratins, laminins, and collagen. Explorations into the regulatory mechanisms underlying epidermal genes have underscored the importance of transcription factors AP-1 and AP-2, retinoic acid receptors, and POU proteins. Transgenic and gene ablation experiments on TGF-alpha and TGF-beta genes have yielded clues as to how the epidermis maintains a balance of growing and differentiating cells.

Animals↗

PubMed1994

Intermediate filaments and disease: mutations that cripple cell strength.

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Animals↗

PubMed1994

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.

Amino Acid Sequence↗

PubMed1994

Genetic bases of epidermolysis bullosa simplex and epidermolytic hyperkeratosis.

Cell Differentiation↗

PubMed1994

Oct-6: a regulator of keratinocyte gene expression in stratified squamous epithelia.

POU domain proteins have been implicated as regulators of differentiation and development, particularly in early embryogenesis and in neural morphogenesis. Given that neural and epidermal lineages originate from a common precursor (ectodermal) cell, we explored the possibility that POU proteins are involved in epidermal differentiation. Using reverse transcription-PCR and degenerate oligonucleotides, we generated several POU domain cDNAs from cultured human epidermal mRNAs. One of these encoded a sequence identical to the rodent Tst-1/SCIP/Oct-6 POU domain. Subsequently, we isolated a cDNA encoding a 45.3-kDa protein with 98% sequence identity to rat Tst-1/SCIP and 94% identity to mouse Oct-6. This protein bound specifically to the canonical octamer motif, warranting its designation as human Oct-6. By RNase protection assays, by PCR, and by immunoblot analysis, Oct-6 was expressed in cultured epidermal keratinocytes. By in situ hybridization, Oct-6 mRNA was detected not only in epidermis but also a variety of other stratified squamous epithelia and with greater signals than testis, the tissue in which this POU protein was originally discovered. Moreover, Oct-6 exerted a marked and specific negative influence on expression of the K5 and K14 genes, abundantly expressed in most dividing stratified squamous epithelial cells and downregulated as cells commit to terminally differentiate. The repressive effect was complex, but it was not observed with Oct-1, nor was it seen with a truncated Oct-6 missing the POU domain. Taken together, our studies suggest that Oct-6 may play an important role in controlling gene expression in stratified squamous epithelia, including epidermis.

Amino Acid Sequence↗

PubMed1994

Intermediate filaments: structure, dynamics, function, and disease.

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Amino Acid Sequence↗

PubMed1994

Genetic mutations in the K1 and K10 genes of patients with epidermolytic hyperkeratosis. Correlation between location and disease severity.

Epidermolytic hyperkeratosis (EH) is a skin disease caused by mutations in the genes encoding K1 and K10, the differentiation-specific keratins of epidermis. To explore the heterogeneity of mutations and to assess whether a correlation exists between disease severity and the extent to which a mutation interferes with keratin network formation, we determined the genetic bases of four severe incidences of EH and one unusually mild case. Two severe cases have the same mutation, K10-R156:C, at a conserved arginine that we previously showed was mutated to a histidine in two unrelated EH families. An additional severe case has a mutation six residues away, still within the amino end of the alpha-helical rod domain of K10. The other severe case has a mutation in the conserved carboxy end of the K1 rod. In contrast, affected members of the atypically mild family have a mutation just proximal to the conserved carboxy end of the K10 rod. By genetic engineering and gene transfection, we demonstrate that each mutation is functionally responsible for the keratin filament aberrations that are typical of keratinocytes cultured from these patients. Moreover, we show that the mild EH mutation less severely affects filament network formation. Taken together, our studies strengthen the link between filament perturbations, cell fragility, and degeneration.

Amino Acid Sequence↗

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↗