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Biomedical subjects

E Fuchs

Publications and source records attributed to E Fuchs.

At least 271 records · Page 15Linked to original sources
PubMed1989

The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells.

When cells from normal human epidermis and from the human squamous cell carcinoma line SCC-13 were seeded on floating rafts of collagen and fibroblasts, they stratified and underwent terminal differentiation. Although the program of differentiation in SCC-13 cells was morphologically abnormal, the cultures resembled normal epidermal raft cultures by expressing the terminal differentiation-specific keratins, K1/K10, and by restricting their proliferative capacity to the basal-like cells of the population. In addition, the differentiating cells of both normal and SCC-13 raft cultures expressed keratins K6 and K16, which are not normally expressed in epidermis, but are synthesized suprabasally during wound-healing and in various epidermal diseases associated with hyperproliferation. While the behavior of normal and SCC-13 rafts was quite similar when they were cultured over normal medium, significant biochemical differences began to emerge when the cultures were exposed to retinoic acid. Most notably, while the SCC-13 cultures still stratified extensively, they showed a marked inhibition of both abnormal (K6/K16) and normal (K1/K10) differentiation-associated keratins, concomitantly with an overall disappearance of differentiated phenotype. Surprisingly, the reduction in K6/K16 in retinoid-treated SCC-13 cultures was not accompanied by a decrease in cell proliferation. Using immunohistochemistry combined with [3H]thymidine labeling, we demonstrate that while the expression of K6 and K16 are often associated with hyperproliferation, these keratins are only produced in the nondividing, differentiating populations of proliferating cultures. Moreover, since their expression can be suppressed without a corresponding decrease in proliferation, the expression of these keratins cannot be essential to the nature of the hyperproliferative epidermal cell.

Blotting, Northern↗
PubMed1989

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation.

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.

Actin Cytoskeleton↗
PubMed1989

A new look into an old problem: keratins as tools to investigate determination, morphogenesis, and differentiation in skin.

We have investigated keratin and keratin mRNA expression during (1) differentiation of stem cells into epidermis and hair follicles and (2) morphogenesis of follicles. Our results indicate that a type I keratin K14 is expressed early in embryonal basal cells. Subsequently, its expression is elevated in the basal layer of developing epidermis but suppressed in developing matrix cells. This difference represents an early and major biochemical distinction between the two diverging cell types. Moreover, because expression of this keratin is not readily influenced by extracellular regulators or cell culture, it suggests a well-defined and narrow window of development during which an irreversible divergence in basal and matrix cells may take place. In contrast to K14, which is expressed very early in development and coincident with basal epidermal differentiation, a hair-specific type I keratin and its mRNA is expressed late in hair matrix development and well after follicle morphogenesis. Besides providing an additional developmental difference between epidermal and hair matrix cells, the hair-specific keratins provide the first demonstration that keratin expression may be a consequence rather than a cause of cell organization and differentiation.

Animals↗
PubMed1989

Unusual patterns of keratin expression in the overlying epidermis of patients with dermatofibromas: biochemical alterations in the epidermis as a consequence of dermal tumors.

Dermatofibromas are frequently associated with acanthosis of the overlying epidermis. Using monospecific antisera and cRNA probes, we have examined the pattern of expression of keratin and keratin mRNA in the affected epidermis of patients with these dermal tumors. Our studies reveal several abnormalities in keratin expression within the thickened areas of overlying epidermis. In two of 15 patients, we detected K6 and K16, keratins which are frequently associated with epidermal diseases of hyperproliferation but are not present in normal epidermis. In both cases, K6 and K16 were found in suprabasal layers, similar to that seen for psoriasis and squamous cell carcinomas. Expression of K6 and K16 in skin samples from patients with dermatofibromas seemed to be dependent upon how near was the tumor to the overlying epidermis, and possibly upon the degree of cellularity within the tumor mass. A second aberration in keratin expression, and one which did not appear to be linked to K6/K16 expression, was the altered expression of the basal epidermal keratin K14. Expression of this keratin and its mRNA was variable, often extending into multiple suprabasal layers and including both basal-like and squamous-like cells. In contrast to the expression of K6/K16, aberrant expression of K14 was a relatively frequent event, occurring in greater than 70% of the dermatofibroma skin samples examined. These observations provide the first biochemical evidence in support of previous morphologic studies, indicating that alterations in epidermal differentiation can occur as a consequence of dermal skin tumors.

Fibroma↗
PubMed1989

Isolation, sequence, and expression of a human keratin K5 gene: transcriptional regulation of keratins and insights into pairwise control.

The mitotically active basal layers of most stratified squamous epithelia express 10 to 30% of their total protein as keratin. The two keratins specifically expressed in these cells are the type II keratin K5 (58 kilodaltons) and its corresponding partner, type I keratin K14 (50 kilodaltons), both of which are essential for the formation of 8-nm filaments. Dissecting the molecular mechanisms underlying the coordinate regulation of the two keratins is an important first step in understanding epidermal differentiation and in designing promoters that will enable delivery and expression of foreign gene products in stratified squamous epithelia, e.g., skin. Previously, we reported the sequence of the gene encoding human K14 (D. Marchuk, S. McCrohon, and E. Fuchs, Cell 39:491-498, 1984; Marchuk et al., Proc. Natl. Acad. Sci. USA 82:1609-1613, 1985). We have now isolated and characterized the gene encoding human K5. The sequence of the coding portion of this gene matched perfectly with that of a partial K5 cDNA sequence obtained from a cultured human epidermal library (R. Lersch and E. Fuchs, Mol. Cell. Biol. 8:486-493, 1988), and gene transfection studies indicated that the gene is functional. Nuclear runoff experiments demonstrated that the K5 and K14 genes were both transcribed at dramatically higher levels in cultured human epidermal cells than in fibroblasts, indicating that at least part of the regulation of the expression of this keratin pair is at the transcriptional level. When the K5 gene was transfected transiently into NIH 3T3 fibroblasts, foreign expression of the gene caused the appearance of endogenous mouse K14 and the subsequent formation of a keratin filament array in the cells. In this case, transcriptional changes did not appear to be involved in the regulation, suggesting that there may be multiple control mechanisms underlying the pairwise expression of keratins.

Amino Acid Sequence↗
PubMed1989

Chronic CEI alters effect of low Na+ diet in normal and coarcted pups. I. BP, renin, and GFR.

To assess the renin-angiotensin (RA) dependence of evolving neonatally induced coarctation hypertension (NICH) in inbred pups, we randomized sex-matched littermates to a high dose of converting-enzyme inhibitor (CEI:MK-421) or placebo from the time of neonatal aortic banding (coarcted) vs. no banding (control). In phase 1 studies, pups were examined serially on normal diet (Bagby and Fuchs, Hypertension In press.): chronic CEI lowered systolic blood pressure (BP) equally in coarcted and controls and failed to prevent systolic BP excess in coarcted dogs. In phase 2 studies, the same pups were exposed to a low sodium (LS) diet at 4 mo of age, a time when the untreated NICH model exhibits increased forelimb systolic BP. Measurements of systolic BP, RA components, renal function, and extracellular volume (ECV) were made before and serially during 12 days on the LS diet. Responses of coarcted and control pups to the LS diet were similar, with or without CEI, providing no evidence for exaggerated angiotensin II (ANG II) dependence in evolving NICH. Independently of coarctation status, chronic CEI significantly modified RA and renal functional responses to the LS diet: a greater renin rise but abnormal renin substrate fall, thus no rise in ANG I or ANG I generation rate; a greater rise in creatinine and a trend toward a greater fall in glomerular filtration rate (GFR). Despite these findings compatible with sustained ANG II deficit, chronic CEI unexpectedly failed to impair maintenance of systolic BP during a superimposed LS diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Chronic CEI alters effect of low Na+ diet in normal and coarcted pups. II. Na+ and H2O balance.

To assess angiotensin (ANG II) dependence of evolving neonatally induced coarctation hypertension (NICH) in inbred pups, we randomized sex-matched littermates to high-dose converting enzyme inhibitor (CEI: MK-421, 3 mg/kg) or placebo from the time of neonatal aortic banding (coarcted) vs. no banding (control). During phase 1 studies over 4 mo postbanding during ad libitum Na+ intake (Bagby and Fuchs, Hypertension Dallas in press). CEI failed to prevent evolution of proximal blood pressure (BP) excess or to impair renal function. Phase 2 studies examine, in the same pups, responses to low Na+ (LS) diet superimposed on chronic CEI at 4 mo, timed to allow development of BP increase in untreated NICH. The present report details metabolic handling and balances of Na+, K+, and fluid for 3 days before (normal Na+ intake) and daily for 11 days after initiation of LS diet, a companion paper describes BP, renin-angiotensin (RA), and renal functional responses. In no case did metabolic responses of coarcted pups to LS diet differ from those of controls, whether on CEI or placebo, whereas responses to LS diet and to CEI reveal positive findings of independent interest. LS diet induced expected renal and fecal Na+ conservation, no net effect on K+ balance, and, despite unexpected free-water diuresis, mild hyponatremia. Chronic CEI impaired maximal renal (but not fecal) Na+ conservation during LS diet, caused exaggerated free-water diuresis but no change in fluid balance, and thus, with the larger Na+ deficit, accounted for greater hyponatremia. CEI caused no net effect on K+ balance. Results indicate normal renal handling of fluid, Na+, and K+ in evolving NICH and provide no evidence for selective intrarenal RA activation or exaggerated ANG II dependence. Findings also suggest that, during LS diet, ANG II is 1) essential for maximum renal Na+ conservation and normal free-water handling, and 2) not essential for fecal Na+ and water conservation or for maintenance of normal water and K+ balances. Results are also compatible with a CEI-induced thirst stimulation and/or osmotic insensitivity and with functional vasopressin deficiency during LS diet.

Animals↗
PubMed1989

[Urine proteins in primates].

Protein excretion and protein fractions either according to their molecular mass or with immunological techniques were studied in the spontaneous morning urine of 17 primate species. The total protein concentration in most of the species ranges between 0.01 and 0.2 mg/ml. The pronounced proteinuria (4 mg/ml) in some south american species (Callithricidae) seems to be remarkable. By using immunoprecipitation (LC-Partigen plates), albumin could be detected in most species, alpha 1-microglobulin in some, and transferrin in few of the species. After electrophoretic separation on pre-cast 1D-micro- SDS-PAA gradient gels (8-25%, semi-automatic Phast-System) followed by CBB R-350 or silver stain respectively, in most species a protein pattern similar to human urine could be observed. As our results show, urine analysis is a suitable tool for noninvasive investigations in primates.

Albuminuria↗
PubMed1989

Exploring the mechanisms underlying cell type-specific and retinoid-mediated expression of keratins.

Keratins constitute a complex and diverse family of proteins that are expressed in a tissue-specific and differentiation-specific fashion. In some cells, retinoids can influence both keratin expression and cell differentiation. In this study of the molecular mechanisms underlying the differential expression of keratins, we examined keratin proteins, mRNAs, and nascent transcripts in a variety of cultured human cell types, including epidermal cells and simple epithelial cells. We also investigated intermediate filament gene expression in fibroblasts, which express vimentin rather than keratin. We found that both cell type-specific and retinoid-mediated expression of keratins are regulated at least in part at the transcriptional level in these cells. However, given the kinetics of the retinoid-mediated response, it seems most likely that the keratin gene expression that is regulated by retinoids reflects a secondary rather than primary response.

Cell Differentiation↗
PubMed1989

[Computer-assisted video processing of Phast gels with ELBA].

Referring to one-dimensional micro electrophoresis-gels, produced at the division of nephrology (Klinikum Göttingen) by using the Phast-System (Pharmacia, Freiburg), the ELBA-program for image evaluation of electrophoretic gels (interactive systems, Heidelberg) was investigated. The digital image, obtained with a computer-controlled video-camera, can be stored up in a personal computer. Independend of time, the digitized data may be retrieved and utilized for evaluation of specific urinary protein patterns. Changes in intensity and constellation of the protein patterns, which may occur during the progress of a disease, can be figured directly in one graphical plot. Therefore, any urinary protein pattern of one or a number of gels can be exactly compared to each other in a short time.

Blood Proteins↗
PubMed1988

Only one out of the three strong ribosomal binding sites of the early region of bacteriophage T7 exhibits high translational efficiency in fragments of about 30 base pairs.

Within the early region of bacteriophage T7 three genes, 0.3, 1 and 1.3, are most efficiently expressed. They belong to the strongest initiation signals of Escherichia coli. In the T7 wild-type situation the proteins are produced with a molar ratio of gene 1:1.3:0.3 protein = 1:3.9:9.7. DNA fragments of about 30 base pairs comprising the ribosomal binding sites (RBS) of these genes were synthesized and cloned into derivatives of the pDS1 vector ribosomal binding sites (RBS) of these genes were synthesized and cloned into two derivatives of the pDS1 vector just upstream of the mouse dihydrofolate reductase gene. Although all tested RBS fragments contained an initiation triplet, a Shine-Dalgarno sequence and some nucleotides upstream and downstream of this region, only the gene 1.3 RBS fragment showed high efficiency whereas those of genes 0.3 and 1 were at the border of significance. The amount of synthesized mRNA was about the same for all three constructs. A major influence of vector-derived sequences on the RBS activity could be ruled out. The high translational activity of the short 1.3 gene RBS seems to be largely due to its primary structure. The other two RBSs studied require much longer sequences for high activity.

Base Composition↗
PubMed1988

Binding sites of atrial natriuretic peptide in human renal tissue-quantification by in vitro receptor autoradiography.

Specific binding sites for atrial natriuretic peptide (99-126) in different areas of normal human renal tissue were quantified by in vitro autoradiography. Our data represent the first characterization of ANP binding sites in different structures of the human kidney. Characterization of ANP binding revealed by Scatchard plot analysis a single class of high affinity binding sites in the glomeruli (Kd 0.53 +/- 0.11 nM; BMax 74.4 +/- 17.86 fmol/mg protein), the vasculature (Kd 0.18 +/- 0.014 nM; BMax 91.6 +/- 25.02 fmol/mg protein), and the medulla (Kd 0.34 +/- 0.13 nM; BMax 106.0 +/- 30.61 fmol/mg protein). These sites may play a key role in the actions of the cardiac hormone in human kidney and in the ameliorating effects of ANP in the recovery from acute renal failure.

Aged↗
PubMed1988

The corticosterone receptive system in the brain of Tupaia belangeri visualized by in vivo autoradiography.

The present investigation deals with in vivo binding of 3H-corticosterone in the brains of tree shrews as visualized by autoradiography. Tree shrews were injected with 3H-corticosterone and brain sections were mounted on slides which were subsequently exposed on tritium sensitive film. The relative labeling of 20 different brain structures was determined densitometrically. The indusium griseum, which demonstrated the highest binding for corticosterone of all brain regions in the autoradiograms, was taken as reference and defined as 100% relative labeling (RL). As in other species, the hippocampal subdivisions of the tree shrew retained high amounts of the steroid (60 to 80% RL). In other parts of the limbic system, medium labeling intensities were observed with approximately 40% RL in the lateral septum. The amygdala was less intensely labeled revealing around 30% RL in the basal accessory, the cortical, central, and the lateral nuclei. Autoradiographic grey values in the ventral striatum and pallidum were comparable to those in the amygdala, but in the islands of Callejae they were approximately as high as in the lateral septum (44% RL). In contrast to previous reports dealing with other species, the tree shrew cerebellum also demonstrated a high binding capacity for corticosterone. The RL was nearly 60% in the cerebellar granular layer. This finding may indicate that the cerebellum also plays a role in mediating the effects of corticosterone in the central nervous system.

Animals↗
PubMed1988

Locomotor activity registration by passive infrared detection in saddle back tamarins and tree shrews.

Locomotor activities of tamarins and tree shrews were monitored using a passive infrared detector (PID) and a small hand-held computer as a recording unit. It is a low-cost system, easy to install and to handle without any disturbing effects on the animals investigated. Recording activity is not limited to specific sites in the cage. A temporal resolution of about two seconds for activity detection will meet most experimental requirements. The method was validated by a comparison of PID-recorded signals with simultaneous behavioral observations using a video-recorder. PID is highly specific, detecting only those movements which involve a change in location. The recording system can be adapted to various experimental conditions or other mammalian species.

Animals↗