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W W Minuth

Publications and source records attributed to W W Minuth.

72 records · Page 4Linked to original sources
PubMed1983

Glycoprotein synthesis of renal collecting duct epithelium cultured as globular bodies.

Cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal calf serum. Within 24 h the explants formed 'globular bodies' which were completely covered by a monolayered epithelium. The cells were differentiated and resembled collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine, a monolayer of collecting duct cells was obtained and used for control experiments. For analysis and identification of synthesized glycoproteins, the globular bodies were incubated with various labelled carbohydrates and amino acids, and then fractionated. Glycoproteins secreted into the culture medium were not detected. Cell-associated glycoproteins were found in crude membrane fractions and then extracted with Triton X-100 for column chromatography, SDS-polyacrylamide electrophoresis in 6 M urea, isoelectrofocusing, and two-dimensional electrophoresis. Two prominent glycoproteins containing galactose and glucosamine were synthesized during the spreading of the epithelium, with an apparent molecular weight of 150,000 and 85,000 (SDS-PAGE). The synthesized glycoproteins differ in their content of radioactive glycoprotein precursor and leucine. The 85,000 dalton monomer glycoprotein has an isoelectric point of 3.5 and was identified by two-dimensional electrophoresis.

Animals↗
PubMed1983

Induction and inhibition of outgrowth and development of renal collecting duct epithelium.

Renal cortical explants consisting of capsula fibrosa with an adherent thin layer of collecting duct anlagen, S-shaped bodies, and nephrogenic blastema were isolated from newborn New Zealand rabbits and cultured in Dulbecco's Modified Eagle's medium for 24 hours. Within this period of time, the explants formed globular bodies surrounded by an epithelium of differentiated collecting duct cells. The outgrowth of the collecting duct cells and the formation of the epithelium occurred only when serum was added to the cultivation medium. Different types and different concentrations of serum were tested; fetal bovine serum and newborn and adult rabbit sera at concentrations of at least 5% induced the outgrowth and spreading of the cells. The surrounding epithelium did not develop in the absence of serum. The outgrowth of the collecting duct cells in serum-supplemented cultivation media was arrested by inhibitors of protein and glycoprotein synthesis and by cytoskeletonal-blocking agents such as cycloheximide (1 x 10(-6) M), actinomycin C1 (2 micrograms/ml), tunica-mycin (1 microgram/ml), 6-diazo-5-oxo-norleucine (2 x 10(-5) M), vinblastine (5 x 10(-6) M), colchicine (1 x 10(-3) M), and cytochalasin B (2 micrograms/ml). In contrast, inhibitors of DNA synthesis, e.g., cytosine arabinoside (2.5 x 10(-5) M), mitomycin (1 x 10(-6) M), and hydroxyurea (2.5 x 10(-3) M), had no influence on the outgrowth.

Animals↗
PubMed1982

Culturing of renal collecting duct epithelium as globular bodies.

Thin kidney cortex explants from newborn rabbits, consisting of the capsula fibrosa with an adherent layer of nephrogenic material (s-shaped bodies and collecting duct anlagen) were isolated and cultured for various periods (up to 15 days) by conventional procedures. Within the first 24 h of culturing, it was observed that the explants form globular bodies covered by a monolayer epithelium. The cells are cuboidal or cylindrical, and may, in later stages, consist of two or even more cell layers. The epithelium shows a polar differentiation, with deep tight junctions at the apical poles and lateral intercellular spaces opening to the basal surface. A basal lamina is not consistently developed. The support of the epithelium consists of fibroblasts and degenerating tissue elements of the explants. It can be shown that the epithelium is derived from the anlagen of the collecting ducts. Structurally, the epithelium resembles a collecting duct epithelium. The experimental system is considered as a useful model to study metabolism and function of the renal collecting duct epithelium, as well as developmental problems, such as cell migration, outgrowth, and differentiation.

Animals↗
PubMed1982

Cell associated glycoproteins synthesized by cultured renal tubular cells.

Thin cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's MEM containing 10% fetal bovine serum. Within 24 h the explants formed globular bodies which were completely covered by a monolayered epithelium. The cells show polar differentiation and resemble the renal collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine additionally a monolayer of renal collecting duct cells was obtained. For the study of glycoprotein synthesis the globular bodies and the collecting duct monolayers were incubated with various labelled carbohydrates, protein and collagen precursors and then fractionated into coarse membrane pellets. The synthesized glycoproteins were regained in 600 x g and 12,000 x g coarse membrane fractions and extracted with Triton X 100 buffer for column chromatography and SDS-polyacrylamide electrophoresis in 6 M urea. In addition to a 85,000 d glycoprotein, a carbohydrate rich collagen like protein (apparent molecular weight in column chromatography 200,000 d, in the SDS-polyacrylamide electrophoresis 150,000 d) was found. The 150,000 d glycoprotein incorporates favorably radioactive proline, sulfate, and smaller amounts of lysine, and leucine. Compared to the 85,000 d glycoprotein a double amount of glucosamine and galactose and four fold amount of fucose was detected. The 85,000 d protein has to be ascribed as a usual glycoprotein, in contrast the 150,000 d protein shows an unusual combination of characteristics and has to be considered as a new type of renal glycoprotein.

Animals↗
PubMed1980

The pig yolk sac I. Fine structure of the posthaematopoietic organ.

Yolk sacs from pig embryos ranging between 18 mm and 55 mm in length were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and histochemistry. The organ was no longer present in embryos of 70 mm length. The endoderm proliferates in embryos of about 20 mm length with gland-like endodermal cell columns and finally becomes stratified, representing over 90% of the yolk sac mass. The endodermal cells show a high activity of oxidoreductases and lysosomal enzymes; their luminal surface bears few absorptive specializations. The mesothelium is inert, as judged from its surface ultrastructure, organelle composition and enzyme content. TEM reveals the endodermal cells to be polarized even in stratified areas. They resemble liver parenchymal cells with respect to their basal villi, which are exposed to capillaries with discontinuous or fenestrated endothelium. Giant mitochondria with crystalline inclusions in the mature endodermal cytoplasm are outnumbered by large stacks of the rough ER, which can amount to 60% of the cytoplasm. This conspicuous RER is suspected to be the production site of serum proteins which are discharged into the vascular bed. Close to the time of the organ's regression, an unusual storage of material in terminal buds of the ER was found. Intercellular canaliculi and the endocytic apparatus of the endoderm are thought to serve regression.

Animals↗
PubMed1980

the pig yolk sac II. Analysis of synthesized proteins.

Since previous morphological studies have revealed abundant rough endoplasmic reticulum in the yolk sac endoderm, pig yolk sac explants from 30 day old embryos were incubated for 3-12 h with [3H]-L-leucine in order to study their protein biosynthesis. They were fractionated into a 12,000 X g-pellet, 105,000 X g-pellet, and supernatant. Newly synthesized proteins in these tissue fractions, and proteins discharged into the culture medium, were analysed with the aid of scintillation technique and identified by column chromatography, SDS-polyacrylamide gel electrophoresis with urea, isoelectrofocusing, and 2D-electrophoresis. Most of the radioactivity incorporated into the tissue fractions was regained from the coarse pellet and was located in the molecular weight region between 70,000 and 45,000 daltons, indicating that most of the newly synthesized proteins are membrane bound and include albumin. Albumin, an acid protein of a MW around 80,000 daltons, and many neutral and basic peptides were present in the culture medium. The yolk sac endodermal cells of the pig synthesize less proteins than those of the cat, although the pig cells display much larger amounts of endoplasmic reticulum.

Animals↗
PubMed1980

Synthesis of serum proteins by the posthaematopoietic feline yolk sac.

The feline yolk sac persists even after the end of its haematopoietic phase with prominent ER-cisternae in the endoderm suggesting biosynthetic capacity. Therefore, yolk sac explants from the 54th day and 57th day were incubated with [3H]-L-leucine in order to study its protein biosynthesis. Newly synthesized proteins were discovered in sliced SDS-polyacrylamide gels by the use of scintillation technique and identified by molecular weight determination and isoelectrofocusing, also using stained electropherograms of unlabeled tissue, serum, and marker proteins. The highest radioactive incorporations were found in 69,000--70,000 dalton proteins and interpreted as serum albumin and alpha-fetoprotein. The autoradiography revealed that the cytoplasm of the endoderm is the site of the most active biosynthesis of proteins which were obviously stored in the ER-cisternae for some time. The yolk sac fluid proteins are almost exclusively serum proteins, although in a very low concentration. We regard a large-scale formation of serum proteins in the yolk sac endoderm as the cause of this organ's very late regression in the cat.

Animals↗
PubMed1980

Newly synthesized proteins in rat glomerular fractions.

Isolated glomerular preparations are known for their capability to synthesize proteins. A thorough biochemical separation and characterization of newly synthesized proteins, however, is lacking. In this study isolated glomeruli from rat kidney (enriched by sieving technique alone or by a combination of sieving and subsequent further enrichment by either sucrose or Ficoll discontinuous centrifugation) were incubated for various periods with protein and glycoprotein precursors. The synthesis of soluble and membrane-bound proteins was studied. Separation of glomerular proteins was carried out by means of SDS-electrophoresis in 6 M urea. The radioactivity of newly synthesized proteins was determined by the scintillation technique. In the leucine-incubated glomeruli, a broad spectrum of newly synthesized monomer proteins ranging from 90,000 to 25,000 daltons was detected, whereas in the glucosamine and galactose incubated glomeruli synthesis of glycoproteins of molecular weights ranging mainly between 90,000 and 60,000 daltons could be observed. Most of the radioactivity incorporated into isolated glomeruli was regained in 600 g (containing basement membrane with residual mesangial/endothelial nuclei and cellular debris) and 12,000 g (membrane structures and mitochondria) pellets, indicating that most of the newly synthesized proteins are membrane bound. A component of newly synthesized glycoprotein (72,000 daltons) could be extracted by mild NaCl treatment and is partly soluble in desoxycholate. Further experiments utilizing isoelectrofocusing and two-dimensional electrophoresis are in progress.

Animals↗
PubMed1978

Mesodermalization of amphibian gastrula ectoderm in transfilter experiments.

Transfilter experiments were carried out with competent gastrula ectoderm of Triturus vulgaris and Ambystoma mexicanum as the reacting materials and blastoporal lip, alcohol-treated bone marrow and archenteron roof as the inducing materials, It was shown that a mesodermal inducing substance is able to penetrate Nuclepore filters of different pore sizes (3.0, 1.0, 0.4, 0.2 micrometer). The degree of mesodermal differentiations depends on the reacting materials, the inductors and the filters which were used in the transfilter experiments.

Ambystoma↗
PubMedDate not supplied

Tissue factory: conceptual design of a modular system for the in vitro generation of functional tissues.

Tissue factory is a modular system designed to generate artificial tissues under optimal perfusion culture conditions. The microenvironment within the culture containers can be fine-tuned to meet the physiological needs of individual tissues, so that the generation of differentiated three-dimensional tissue constructs becomes possible. An optimal physiological environment is created by modulating a liquid phase as well as an artificial interstitium surrounding the growing construct. An innovative construction principle allows production of tissue culture containers, gas exchangers, and gas expanders at minimal material expenditure. Therefore it will be possible for the first time to produce sterile one-way perfusion culture modules for the generation of artificial tissues. The modules can be used separately as well as in a combined module. The system is designed to provide a possible platform for the standardized production of artificial tissues for future applications in biomedicine.

Cell Culture Techniques↗
PubMedDate not supplied

Modulation of cell differentiation in perfusion culture.

An in vitro model was used to investigate the terminal differentiation mechanisms leading from embryonic to adult renal tissue. For these experiments the capsula fibrosa with adherent embryonic tissue was isolated from neonatal rabbit kidneys. These explants were mounted onto special tissue carriers and cultured in medium containing serum for 24 h. During that time collecting duct (CD) cells grew out and formed a monolayered epithelium covering the whole surface of the explant. The carriers were then transferred to perfusion culture containers to obtain an optimal degree of differentiation. A special type of container allowed us to continuously superfuse the epithelia with individual media on the luminal and basal sides. Using this method it became possible to culture embryonic CD epithelia in a fluid gradient for weeks. The epithelia were superfused with standard Iscove's modified Dulbecco's medium (IMDM) on the basal side, while IMDM containing additional NaCl was used on the luminal side. In controls IMDM was superfused on both the luminal and basal sides. It was found that the degree of differentiation in the CD epithelia is dependent on the influence of fluid gradient exposure. Perfusion culture under isotonic conditions revealed that less than 5% of cells were immunopositive for principal and intercalated cell features, while epithelia cultured in a luminal-basal gradient showed more than 80% positive cells. Immunoreactivity for characteristic markers started to develop after an unexpectedly long latent period of 3-6 days, then increased continuously during the following 5 days and reached a maximum on day 14. After switching back from the gradient to isotonic culture conditions the immunoreactivity for some markers decreased within 5 days, while other characteristic features remained stable. Thus, differentiation was not only under the control of growth factors but was also regulated by the electrolyte environment.

Animals↗
PubMedDate not supplied

Embryonic renal collecting duct cell differentiation is influenced in a concentration-dependent manner by the electrolyte environment.

BACKGROUND: During kidney development, the embryonic collecting duct (CD) epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. It is unknown by which molecular mechanism the different cell types arise. We have experimental evidence that the electrolyte environment is involved in the process of terminal cell differentiation. METHODS: Embryonic CD epithelia from neonatal rabbit kidneys were microsurgically isolated and maintained in gradient perfusion culture for 13 days under serum-free conditions. Controls were maintained in the same medium (Iscove's modified Dulbecco's medium; IMDM) on basal and luminal sides. Experimental series were performed with IMDM only on the basal side, while on the luminal side IMDM with increasing concentrations of NaCl was used. Finally, the development of principal and intercalated cell features was registered by immunohistochemical labeling with markers specific for adult CD cells. RESULTS: Immunohistochemical markers show that the differentiation pattern is quite different when the embryonic CD epithelia are cultured in IMDM only as compared with specimens kept in IMDM supplemented with 3-24 mmol/l NaCl on the luminal cell side. First signs of changes in development were seen when low doses of 3-6 mmol/l NaCl were added. CONCLUSIONS: We conclude that facultative protein expression in embryonic CD epithelium is influenced by the electrolyte environment and starts to be upregulated after administration of unexpectedly low doses of 3-6 mmol/l NaCl added to the luminal perfusion culture medium and increases in a concentration-dependent manner.

Animals↗
PubMedDate not supplied

Action of aldosterone on citrate synthase in cultured renal collecting duct cells.

Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enzymatic activity of citrate synthase during increase in Na+ transport. Control epithelia showed citrate synthase activity of 71 +/- 3 mU/mg protein (n = 28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 +/- 6 mU/mg at 1 h (n = 5), to 88 +/- 6 mU/mg at 2 h (n = 6) and to 93 +/- 8 mU/mg protein at 3 h (n = 5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.

Adenosine Triphosphate↗
PubMedDate not supplied

Action of aldosterone on protein expression in cultured collecting duct cells from neonatal rabbit kidney.

To investigate whether 'aldosterone-induced proteins' could be detected in mammalian species, cultured renal collecting duct epithelia from neonatal rabbit kidneys were labelled under aldosterone administration with radioactive methionine and subsequently fractionated into cytosolic and coarse membrane protein fractions. Newly synthesized proteins were then analyzed by SDS-PAGE, isoelectric focussing and two-dimensional electrophoresis. Quantitative estimates of individual newly synthesized proteins were performed utilizing gel slicing, scintillation counting and autoradiography. The labelling experiments demonstrated that, in comparison to controls, aldosterone (1 X 10(-6) M) generally increased the amount of radioactive protein. No qualitative changes in the pattern of newly synthesized proteins and, therefore, no classical aldosterone-induced proteins were observed. The increase of radioactive protein was already seen after 1, 6, and 18 h of hormone treatment. The effect could be blocked partially by spironolactone (1.5 X 10(-4) M), and totally by amiloride (1 X 10(-6) M), g-strophantin (5 X 10(-4) M), and cycloheximide (1 X 10(-6) M. Thus, the interference of aldosterone action at the receptor level, the Na+ channels and the Na+/K(+)-ATPase pump demonstrate that the expression of proteins in cultured renal collecting duct cells is a sensitive system and seems to be controlled by aldosterone at the receptor level, but also counter-controlled by specific plasma membrane sites.

Aldosterone↗
PubMedDate not supplied

Action of aldosterone on cultured renal collecting duct cells during the latent period.

The effects of aldosterone on protein synthesis in the latent period were investigated on cultured renal collecting duct cells from neonatal rabbit kidneys. Tissue was incubated with radioactively labelled uridine and amino acids and then precipitated with trichloroacetic acid in order to determine the intracellular precursor pool and identify new synthesis of RNA and protein. During the latent period, aldosterone increased the intracellular radioactive uridine pool and total radioactive RNA content already 20 and 60 min after its application; conversely 40 min after aldosterone introduction, no stimulation was found. Further experiments revealed that the intracellular radioactive amino acid pool was generally increased by aldosterone after 20, 40 and 60 min, while a distinct increased radioactive protein content was found to be induced by aldosterone only after 40 min. This indicates that aldosterone increases the uptake of RNA and protein precursors and the new synthesis of RNA and proteins. These events seem to to be regulated not continuously but intermittently. The induced proteins possibly take part in the mediation of the early hormone response. Experiments with the aldosterone antagonist, spironolactone, provide evidence for the specificity of the described hormone effects. The results after application of the Na+ channel blocker, amiloride, and the Na+/K(+)-ATPase inhibitor, G-strophanthin, indicate that the aldosterone effects are controlled by Na+ channels and Na+ pumps and therefore by the intracellular Na+ content. The inhibitory effect of cycloheximide on the aldosterone-induced protein synthesis indicates the role of these proteins on the hormone-stimulated Na+ transport.

Aldosterone↗
PubMedDate not supplied

Electrolyte environment modulates differentiation in embryonic renal collecting duct epithelia.

The influence of electrolytes on the development of renal principal and intercalated collecting duct cells is unknown. Consequently embryonic collecting duct epithelia were exposed to different electrolyte concentrations, and their degree of differentiation was registered by immunohistochemical methods. Embryonic collecting duct epithelia were isolated from neonatal rabbit kidneys and placed on tissue carriers. The apical urine and the basal serum compartments were simulated in a gradient culture container. The two sides of the epithelium were each constantly superfused with medium for 13 days. In controls the medium on both apical and basal side was standard Iscove's modified Dulbecco's Medium (IMDM) with 112 mmol/l Na+ and 85 mmol/l Cl-. In experimental series the NaCl concentration at the basal side of the epithelium was increased up to 137 mmol/l Na+ and 99 mmol/l Cl- as found in the serum of neonatal rabbits. Light microscopy revealed morphologically faultless epithelia following gradient perfusion culture in standard and NaCl-adapted IMDM. The development of principal and intercalated cell features was monitored with the monoclonal antibodies 703, 503, PCD9, and peanut lectin. Cells immunopositive for monoclonal antibody 703, for example, increased from less than 10% in controls to more than 80% in NaCl-adapted IMDM. It is a new finding that the development of collecting duct cell features is influenced by the extracellular electrolyte environment.

Animals↗