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K D Spindler

Publications and source records attributed to K D Spindler.

At least 37 records · Page 2Linked to original sources
PubMed1996

A muscarinic acetylcholine receptor, present in the epithelial cell line from Chironomus tentans.

A muscarinic cholinergic receptor (mAchR) is present in the non-neuronal epithelial cell line from Chironomus tentans. Scatchard plot analysis (KD = 1.4 nM) using the non-selective antagonist quinuclidinylbenzilate (QNB), as well as kinetic data (KD = 1.7 nM), reveals one class of high affinity binding sites. About 2000 binding sites/cell are present. The receptor concentration (54.5 +/- 7.5 fmol/mg protein) is comparable to the values reported from insect brain. The receptor interacts only with muscarinic ligands; nicotinergic acetylcholine receptors are not present. Binding properties are not comparable to any of the muscarinic subtypes known from vertebrate tissues. The rank order of competition of radiolabelled QNB is: QNB > atropine > PrBCM > oxotremorine, pirenzepine > methoctramine. The competition curve obtained with carbamylcholine is shifted to higher ligand concentrations in the presence of Gpp(NH)p and to lower carbamylcholine concentrations by 1 mM NEM. With antibodies against muscarinic receptor from calf brain one band with a molecular weight of about 80 kDa is detected on Western blots. The moulting hormone 20-OH-ecdysone transiently increases the concentration of muscarinic receptor.

Animals↗
PubMed1995

Immunological studies on the developmental and chromosomal distribution of ecdysteroid receptor protein in Chironomus tentans.

Antisera were raised against different domains of a putative ecdysteroid receptor (cEcRH) of Chironomus tentans. All the antisera reacted with a 68,000 dalton protein exhibiting DNA binding properties. Additionally, we were able to demonstrate that the antisera immunoprecipitate protein which binds a radioactively labeled ecdysteroid (Ec), i.e., [3H]ponasterone A, with high specificity. These properties indicate that the antisera recognize specifically an endogenous ecdysteroid receptor protein (cEcR) in C. tentans cells and thus are suitable for the following quantitative and qualitative immunological and immunohistochemical investigations. The cellular level of cEcR varies during development, and it is particularly low in oligopausing larvae. In polytene chromosomes of prepupal salivary glands, cEcR is located at approximately 50 transcriptionally active loci. These loci include both early ecdysteroid (Ec)-inducible puff sites, such as the locus containing the gene coding for the homolog of the E75 protein in Drosophila melanogaster, as well as late Ec-inducible puff-sites. The latter group comprises a locus of a gene specifying the homolog of the D. melanogaster ultraspiracle protein. However, loci of genes coding for salivary gland secretory proteins (e.g., Balbiani ring forming chromosome regions) do not specifically react with the antisera. Thus, the developmental regulation of these genes is not directly controlled by Ec. Polytene chromosomes of oligopausing larvae show hardly any loci that contain cEcR. The few detected correspond, with few exceptions, to the most potent cEcR binding sites found in prepupae.

Amino Acid Sequence↗
PubMed1995

Characterization of N-linked carbohydrate chains of the crayfish, Astacus leptodactylus hemocyanin.

The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and N-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzymatic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH2, the subfractions were characterized by 1H NMR spectroscopy. A total of six oligosaccharides, ranging from Man4GlcNAc2 to Man9GlcNAc2 is present, Man6GlcNAc2 representing the most abundant one with 57% of all oligosaccharides.

Animals↗
PubMed1992

Protein production and the molting cycle in the crayfish Astacus leptodactylus (Nordmann, 1842). II. Hemocyanin and protein synthesis in the midgut gland.

The midgut gland from the crayfish Astacus leptodactylus synthesizes and secretes hemocyanin and proteins linearly for at least 24 hr in vitro. There are no sex-specific differences in the rate of protein and hemocyanin synthesis. During a standard incubation of 5 hr, up to 64% of the newly synthesized protein represents hemocyanin, which is also the predominant secretory protein. There are pronounced changes in the rate of hemocyanin synthesis during the molting cycle which coincide with corresponding changes in total protein synthesis. The titer of protein and hemocyanin synthesis during an intermolt phase exhibits a biphasic profile with the highest values in intermolt stage C4 and in premolt stage D1.

Animals↗
PubMed1991

Demonstration of beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-hexosaminidase in Drosophila Kc-cells.

Kc-cells from Drosophila melanogaster, grown under serum-free conditions, produce two beta-hexosaminidases and secrete these enzymes into the medium. The two enzymes were separated by DEAE-exchange chromatography. According to their substrate specificities one enzyme is a beta-N-acetyl-D-glucosaminidase (E.C.3.2.1.30), the other one a beta-N-acetyl-D-hexosaminidase (E.C.3.2.1.52). The beta-N-acetyl-D-glucosaminidase is predominant in the medium, the beta-N-acetyl-D-hexosaminidase within the cells. The Km values for the substrates pNP-GlcNAc, pNP-GalNAc, and (GlcNAc)2 are 0.8, 16.73, and 1.67 mM for the beta-N-acetyl-D-glucosaminidase and 0.24, 0.44, and 0.2 mM for the beta-N-acetyl-D-hexosaminidase. Both enzymes are inhibited by the products and the beta-N-acetyl-D-glucosaminidase is also inhibited stereospecifically by the substrates pNP-GlcNAc and (GlcNAc)2. Both enzymes are inhibited in a partial competitive way by acetamidolactones, the Kis being as low as 0.1 microM.

Acetylglucosaminidase↗
PubMed1991

Physical properties of beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-hexosaminidase from Drosophila Kc-cells.

Kc-cells from Drosophila produce two different beta-D-hexosaminidases, a beta-N-acetyl-D-glucosaminidase (E.C.3.2.1.30) and a beta-N-acetyl-D-hexosaminidase (E.C.3.2.1.52), which are also secreted into the medium. The Mr of both enzymes is about 126,000 +/- 9,700; the S-values are 8.37 +/- 0.44. Both enzymes have about the same pH optima at 5.5 and the same thermal stability. The temperature optima are identical (50 degrees C) for both enzymes if p-nitrophenyl-N-acetylglucosaminide is used as a substrate. However, when p-nitrophenyl-N-acetylgalactosaminide is used as the substrate the beta-N-acetyl-D-hexosaminidase has a temperature optimum about 10 degrees C higher. With higher salt concentrations, the activity of the beta-N-acetyl-D-glucosaminidase increases, whereas beta-N-acetyl-D-hexosaminidase is inhibited. Both enzymes also differ in their sensitivity to urea, the beta-N-acetyl-D-hexosaminidase being less sensitive than the beta-N-acetyl-D-glucosaminidase.

Acetylglucosaminidase↗
PubMed1991

Electron microscopic demonstration of glucocorticoid recognition sites on isolated rat hepatocytes.

Ultrastructural evidence is presented for the presence of membrane-bound glucocorticoid recognition and binding sites. Corticosterone was derivatized at 3 different positions and coupled covalently to bovine serum albumin (BSA). All three derivatives competed for binding of [3H]corticosterone by isolated rat hepatocytes. The most effective competitor, corticosterone-succinate-BSA (CSB), was adsorbed onto colloidal gold particles (CSB-gold, 17 +/- 3 nm dia). When isolated rat hepatocytes or mouse pituitary tumor cells (AtT 20) are incubated with CSB-gold, specific binding in the microvilli-rich region of these cells is seen. This binding of CSB-gold is reduced by about 50% in the presence of unlabelled CSB or corticosterone.

Animals↗
PubMed1991

Uptake of corticosterone into isolated rat liver cells: possible involvement of Na+/K(+)-ATPase.

Isolated rat hepatocytes possess a saturable glucocorticoid uptake system with high affinity (Kd value = 2.8 +/- 0.7 x 10(-8) M; 318,000 +/- 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30-40% if the cells are incubated simultaneously with [3H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na+/K(+)-ATPase-inhibitors, ouabain and quercetine. These Na+/K(+)-ATPase-blockers exert half-maximal inhibition at 3 x 10(-7) and 3 x 10(-6) M, respectively. A slight increase in K+ concentration and a corresponding decrease in Na+ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5 alpha- and 5 beta-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.

2,4-Dinitrophenol↗
PubMed1991

Opioid receptor proteins in human brain. Detection with monoclonal anti-idiotypic antibodies.

In order to identify opioid receptor proteins, we first ascertained the presence of mu-receptors in membranes of the human cerebral frontal cortex, using binding studies performed with sufentanil. Sufentanil binding was reversed by naloxone and prevented by an anti-idiotypic opioid receptor-specific antibody. This antibody was bound specifically by proteins (molecular weights of 66 and 68 Kda) detected in frontal cortex membrane preparations by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis in combination with western blot analysis. Binding of the same antibody was achieved in dot blots of homologous cerebral and cerebellar cortex preparations.

Antibodies, Anti-Idiotypic↗
PubMed1990

Structural and developmental analysis of a gene cloned from the early ecdysterone-inducible puff site, I-18C, in Chironomus tentans.

A gene (I-18C) cloned from the early ecdysterone (EDS)-inducible puff site, I-18C, in Chironomus tentans salivary glands, codes by differential processing for at least five transcripts which can be grouped into spliced and unspliced transcripts. The spliced group comprises several RNAs of approx. 4.6 kb, and the unspliced group comprises the 1.8-kb and 6.5-kb RNAs. All these transcripts have a similar or the same transcription start point. The steady-state level of the spliced 4.6-kb RNAs reveals some parallels with EDS concentrations in animals during the various developmental stages analyzed, while the levels of the unspliced 1.8-kb and 6.5-kb RNAs do not correlate with the EDS concentrations.

Animals↗
PubMed1990

Chitin metabolism: a target for drugs against parasites.

Chitin is an important component of the exoskeleton of arthropods and of the egg shell in nematodes, but it does not occur in vertebrates. Therefore, it represents a useful target for drugs against ectoparasitic crustaceans, insects and endoparasitic nematodes. In this review we describe the basic characteristics of chitin, chitin synthesis and degradation and the hormonal regulation of chitin metabolism. Substances interfering with chitin metabolism like benzoylphenyl-urea derivatives but also some recently detected compounds are described. The necessity for a more detailed understanding of chitin metabolism and the establishment of better model systems, like e.g. chitin producing insect cell lines, is stressed and some examples are given in this review.

Animals↗
PubMed1987

20 (OH) ecdysone-induced transition from intermolt to premolt protein biosynthesis patterns in the hypodermis of the crayfish, Astacus leptodactylus, in vitro.

20 (OH) Ecdysone-induced alteration of protein biosynthesis and secretion by the hypodermis of the crayfish Astacus leptodactylus was studied using isolated tissues in vitro. Characteristic quantitative and qualitative changes of late intermolt tissues are governed by the hormone in a dose- and time-dependent manner. The greatest response to the hormone was observed in tissues which were exposed for 48 hr to 10(-8) M 20 (OH) ecdysone, followed by 1 day of hormone withdrawal. The observed differences correspond to the reprogramming in protein biosynthesis after transition from intermolt to the premolt stage.

Animals↗
PubMed1987

Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea Decapoda): quantitative and qualitative changes.

During the embryogenesis of Palaemon serratus profound changes in ecdysteroid concentrations were found. Ecdysteroid concentrations increase at the appearance of the Y-organ and a slight decrease is observed just before hatching. Two peaks of ecdysteroid concentration occur between these two events if Palaemon is reared at 19 degrees, but only one broad maximum is observed after rearing at 11-12 degrees. The pattern of ecdysteroids changes during embryogenesis; the ecdysone level decreases, while the 20-OH-ecdysone concentration and the amount of high polarity products increase. During the embryonic stage C the ratio of 20-OH-ecdysone to ecdysone was shown by HPLC analysis to steadily increase. After enzymatic hydrolysis of high-polarity products the main RIA-positive material elutes from reversed-phase HPLC columns as 20-OH-ecdysone.

Animals↗
PubMed1986

Ecdysteroid levels during the larval development of the spider crab Hyas araneus.

Ecdysteroid levels were determined by radioimmunoassay during the larval development of the spider crab Hyas araneus L. In each of the three larval instars (zoea I, zoea II, megalopa) a minimum in ecdysteroid levels was found during early postmoult and a maximum during premoult. The amounts of ecdysteroids increased during development and reached maxima of 93, 137, and 165 pg ecdysone equivalents per larva in zoea I, zoea II, and megalopa, respectively. Ecdysteroid concentrations per unit of body dry weight or carbon increased during each moulting cycle, but the average values showed decreasing tendency from the first to the last instar. The main ecdysteroid in all larval instars and during the moulting cycle was 20-OH-ecdysone, and practically no ecdysone could be detected by HPLC-RIA.

Animals↗