Biomedical subjects
E Fuchs
Publications and source records attributed to E Fuchs.
Elucidating the early stages of keratin filament assembly.
Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.
Epidermal differentiation: the bare essentials.
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Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.
To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.
TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.
In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.
Regulation of a human epidermal keratin gene: sequences and nuclear factors involved in keratinocyte-specific transcription.
The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assays to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5'-GCCTGCAGGC-3', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.
Activation of glycogenolysis by stimulation of the hepatic nerves in perfused livers of guinea pig and tree shrew as compared to rat: differences in the mode of action.
A study on the metabolic and hemodynamic actions of hepatic nerve stimulation in the perfused liver of guinea pig and tree shrew as compared to rat was performed, since the density of liver innervation was reported to be different. 1) Nerve stimulation resulted in an increase in glucose release and decrease in lactate uptake or in a shift to output as well as a decrease in portal flow in all three species. The change in glucose output was very similar, that in lactate balance and flow was smaller in tree shrew than in guinea pig and rat. Apparently, the metabolic and hemodynamic changes did not reflect the different densities of liver innervation. 2) The overflow of the neurotransmitter noradrenaline into the hepatic vein differed very clearly in the three animals. In the guinea pig and tree shrew the maximal increase in noradrenaline concentration measured in the effluent was about 6-7-fold higher than in the rat. 3) The content of noradrenaline in the liver in vivo was about five-fold higher in the guinea pig and again another four-fold higher in the tree shrew than in the rat. The contents of adrenaline and dopamine were very low in comparison to those of noradrenaline. The different hepatic noradrenaline contents of the three species investigated are in line with the anatomical findings on the different innervation density. 4) Inhibitors of eicosanoid synthesis reduced the nerve stimulation-dependent metabolic and hemodynamic alterations in guinea pig liver as in rat liver indicating a similar mechanism in these species. Apparently, prostaglandins might be involved as mediators or modulators of nerve actions also in the more densely innervated guinea pig liver and not only in the less densely innervated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)
Ultrastructural and histochemical observations on secretory phenomena in the resting human mammary gland.
Ultrastructure and histochemistry of clinically normal appearing tissue and secretions of non-lactating human mammary glands have been investigated in order to document and analyse secretory phenomena in the resting gland. The material studied originated from women of different ages (18-74 years) who underwent plastic surgery or surgery for various disorders of the breast. The epithelia of small ducts and of alveolar enlargements as well as acini contained moderate amounts of mitochondria and of cisterns of the rough endoplasmic reticulum; the transcisterns of the Golgi apparatus which were surrounded by smooth and coated vesicles, exhibited modest dilatations, the number of lysosomes increased with age; regularly glycogen particles and bundles of intracellular filaments (phi 5 nm) were to be observed. Typical casein vesicles and stages of apocrine secretion of milk fat globules were not seen. The following features indicated secretory activity: differently sized vesicles and granules with flocculent, dense, or light contents were regularly to be seen in the apical cytoplasm often immediately below the apical plasma membrane of the epithelia of the small ducts and even more frequently in the alveolar enlargements. Secretory products of fine granular or filamentous structure, probably containing proteins, were frequently found within the lumen. Different types of lipid and liposome-like particles were detected both in intracellular localization as well as in ductal lumina. As demonstrated by lectin histochemistry the secretory products also contained a considerable amount of carbohydrate components. The composition of the secretory products of the resting gland is of clinical interest since the chronical deposition of secretions, which among others possibly contain enzymes producing oxygen radicals, may lead to pathological changes of mammary gland tissue.
Histaminergic system in the tree shrew brain.
This study mapped the histamine-immunoreactive neuronal system in the brain of the tree shrew (Tupaia belangeri) and compared its structure with that of the rat and guinea pig. The histamine-containing cell bodies lay in the posterior ventral hypothalamus in the tuberomammillary complex, as in the rodents. The morphology of this complex resembled that of the rat. The histaminergic axons projected to nearly all parts of the brain. The main ascending bundle ran ventromedially: the densest innervation was found in the ventral hypothalamus, preoptic area, septum, medial part of nucleus accumbens, and bed nucleus of the stria terminalis. High fiber densities were present in the amygdaloid nuclei and claustrum. Another pathway ran dorsomedially along the periventricular hypothalamus and sent fibers to all parts of the diencephalon. Part of these fibers followed the central gray to the midbrain and spread laterally below the inferior colliculus. Another descending pathway ran through the interfascicular and medial raphe nuclei to meet the pontine central gray. The densest fiber networks were seen in the dorsal tegmental and parabrachial nuclei, and around the locus coeruleus. Also the substantia nigra, interpeduncular and mesencephalic reticular nuclei, colliculi, and vestibular and raphe nuclei received a dense histaminergic innervation. The organization of the fibers in the tree shrew brain resembled more that in the guinea pig than that in the rat. As compared with the guinea pig, more fibers were present, particularly in the globus pallidus, central thalamus, and deep cerebellar nuclei. No fibers were seen in the outer layer of the piriform cortex. In Tupaia, a laminar organization of the fibers was evident in the hippocampus, in contrast to the rodents. Also, a dense periventricular fiber plexus was prominent.
Serum immunoreactive erythropoietin in HIV-infected patients.
Serum immunoreactive erythropoietin (SIE) and hemoglobin levels were measured in 152 patients infected with the human immunodeficiency virus. Anemia was present in 18% of asymptomatic patients who tested positive for the human immunodeficiency virus, 50% of patients with a condition related to the acquired immunodeficiency syndrome (AIDS), and 75% of patients with AIDS. The mean SIE level for untreated AIDS patients (26.2 +/- 2.4 mU/mL) was greater than for patients who tested positive for human immunodeficiency virus or patients with an AIDS-related condition but not outside the normal range for SIE (4 to 26 mU/mL), and the incremental increase in SIE level for a given decline in hemoglobin level was much less in AIDS patients than in patients with uncomplicated iron deficiency anemia. Forty-two patients were treated with zidovudine, and the hemoglobin level fell 10 g/L or more in 48%. In contrast to the untreated patients, however, the mean SIE level rose 10-fold to 214 mU/mL, and the incremental change in SIE level for a given decline in hemoglobin level was markedly increased. In the zidovudine-treated patients, erythrocyte mean corpuscular volume also rose significantly from a mean of 88.1 fL to 102 fL. However, in only 1 patient was there a corresponding increase in reticulocytes and in none was there amelioration of anemia. The data indicate that SIE level is inappropriately low in anemic AIDS patients. The ability of these patients to produce erythropoietin is intact and can be expressed with zidovudine therapy. However, even very high levels of SIE fail to stimulate erythropoiesis adequately.
Histamine-immunoreactive nerve fibers in the mammalian spinal cord.
New sensitive antisera against histamine were used to study the distribution of histamine-immunoreactive nerve fibers in the spinal cord of several mammalian species. Tissues were fixed with carbodiimide by transcardiac perfusion or immersion. A few immunoreactive nerve fibers were found in the cervical spinal cord of the rat in the superficial laminae of the dorsal horn, around the central canal and scattered in the anterior horn. The density of immunoreactive fibers in the cervical spinal cord of the guinea pig and tree shrew was higher, but still low. The densest networks of histamine-immunoreactive fibers were seen in the cervical spinal cord of the pig. The laminar distribution of histamine-immunoreactive fibers was similar in all species. Histamine-immunoreactive fibers were densest in lamina X, followed by laminae I-II. Scattered fibers were also seen in the white matter in the lateral and posterior funiculus in the pig. In the rat and the guinea pig, no histamine-immunoreactive cell bodies were seen in the spinal sensory ganglia. The results suggest that the histamine-immunoreactive nerve fibers in the spinal cord may originate from the brain, probably from the posterior hypothalamus, and the fiber projection is more extensive in higher mammalian species. The role of histamine in the spinal cord is not known, but it may be involved in, e.g., pain sensation.
[Ethics concerning the fetus].
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Effect of atrial natriuretic factor (ANF) on nicotinic acetylcholine receptor channels in bovine chromaffin cells.
Bovine chromaffin cells have binding sites for rat atrial natriuretic factor (ANF), as demonstrated autoradiographically by using the 125I-labelled peptide. Patch-clamp recording revealed that ANF reduces acetylcholine (ACh)-induced membrane currents in chromaffin cells at physiological membrane potentials. The effect was dose-dependent with the IC50 value being 5.2 microM ANF and the Hill coefficient close to 1. The channel block was absent at positive membrane potentials, indicating a non-competitive interaction of ANF with the open ACh receptor channel. Fragments of ANF had a much less pronounced action, which is possible due to their structure and molecular charge being different to ANF. The block of nicotinic ACh receptor channels may enable ANF to control the secretion of catecholamines from adrenal chromaffin cells.
A family of type I keratin genes and the homeobox-2 gene complex are closely linked to the rex locus on mouse chromosome 11.
Type I and type II keratins are major constituents of intermediate filaments that play a fundamental role in the cytoskeletal network. By using both somatic cell hybrids and conventional and interspecific linkage crosses, several genes encoding type I keratins, including the epidermal keratin K10, were shown to be closely linked to the homeobox-2 complex and the rex locus on mouse chromosome 11. The absence of crossovers between type I keratin-encoding genes and rex (N = 239), a locus affecting hair development, raises the possibility that mutations at rex and neighboring loci affecting skin and hair development involve type I keratin genes.
[About the question of indication for surgery of retractile testis].
Histometric studies in 34 boys with retractile testes and 21 controls with normal descent showed no significant statistic differences with regard to tubulus diameter and spermatogonia count. Operative treatment of retractile testes, therefore, is not indicated.
Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.
A construct containing approximately 2500 base pairs (bp) of 5' upstream and approximately 700 bp of 3' downstream sequence was used to drive the expression of an intronless human K14 gene in vitro and in vivo. To track the expression of the gene, a small sequence encoding the antigenic portion of neuropeptide substance P was inserted in frame 5' to the TGA translation stop codon of the gene. Surprisingly, this gene was expressed promiscuously in a wide variety of cultured cells transiently transfected with the construct. In contrast, when introduced into the germ line of transgenic mice, the construct was expressed in a fashion analogous to the endogenous K14 gene--namely, in the basal layer of stratified squamous epithelia. Our results suggest that some regulatory mechanism is overridden as a consequence of transient transfection but that sequences that can control proper K14 expression are present in the construct. The appropriate tissue-specific and differentiation-specific expression of K14.P in transgenic mice is an important first step in characterizing a promoter that could be employed to drive the foreign expression of drug-related genes in the epidermis of skin grafts.
Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
We have deleted cDNA sequences encoding portions of the amino- and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy- and amino-terminal alpha-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.