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K D Spindler

Publications and source records attributed to K D Spindler.

51 records · Page 3Linked to original sources
PubMed1983

Uptake and retention of moulting hormones by the integument of crayfishes in vitro: I. Influence of temperature, hormone concentration and hormone structure.

Uptake kinetics of [3H]ecdysone into crayfish hypodermis in vitro reveals three different components: a very rapid (within 1 min), highly temperature-dependent (Q10 = 3) and high uptake rate, a second much lower uptake which is only slightly temperature-dependent (Q10 = 1-1.3), and a plateau which is reached after about 20 min at 20 degrees C. Preincubation of tissue with unlabelled hormone reduces the initial uptake rate to a value characteristic for the second, less steep uptake phase. The activation energy for the initial uptake is 45 kJ/mole. At equilibrium the hormone is 4-fold enriched as compared to the medium, the delta G for this enrichment being 3 kJ/mole. The efflux is much lower than the influx and is linear. There is no linear relation between external hormone concentration and initial uptake rate. The initial uptake can be significantly reduced by an excess of diverse ecdysteroids with moulting hormone activity but not by hydrocortisone.

Animals↗
PubMed1983

Uptake and retention of moulting hormones by the integument of crayfishes in vitro. II. Influence of metabolic inhibitors and sulphydryl group inhibitors.

Antimycin A and 2,4-dinitrophenol reduced both the initial uptake and the total uptake of ecdysone into crayfish hypodermis in vitro. Antimycin A did not reduce the retention of moulting hormone, in contrast to 2,4-dinitrophenol. The inhibitory effect of antimycin A could be completely overcome. The membrane-mediated ecdysteroid uptake was sensitive to the sulphydryl group inhibitors N-ethylmaleinimide (NEM) and p-chloromercuriphenylsulphonate (PCMPS). Inhibition of steroid uptake by PCMPS was completely reversed by dithiothreitol.

2,4-Dinitrophenol↗
PubMed1982

Characterization of nuclear ecdysteroid receptor from crayfish integument.

The existence of nuclear ecdysteroid receptors in isolated nuclei from crayfish integument was demonstrated. Nuclei were prepared from crayfish hypodermis by use of a modified Chauveau-method and exposed to [3H]-ecdysteroids. The nuclear hormone receptor complex sedimented at about 5 S and the available binding sites had a high affinity to [3H]-ecdysteroids. Treatment of the nuclei with pronase and DNase completed or partially reduced nuclear bound ecdysone, whereas RNase was without any effect. Competition of nuclear bound ecdysone by various steroids confirmed ponasterone A and kaladasterone as the best competitors followed by 20-OH-ecdysone and ecdysone, whereas compounds like poststerone, "triol" or hydrocortisone did not compete. If [3H]-20-OH-ecdysone was used as the radio-ligand, 20-OH-ecdysone was a better competitor than ecdysone indicating that there may be two different binding sites for ecdysone and 20-OH-ecdysone; this was further substantiated by the finding of two different number of binding sites for these steroids.

Animals↗
PubMed1982

Ecdysteroid receptors: a comparison of cytoplasmic and nuclear receptors from crayfish hypodermis.

Cytoplasmic and nuclear ecdysteroid receptors from intermolt Orconectes limosus integument have been compared. The specificities as well as the KD values for three [3H]ecdysteroids are very similar in both preparations. Using a photoaffinity labelling technique, the molecular mass of the hormone-receptor complexes from both preparations was determined by sodium dodecyl sulfate-gel electrophoresis as 70 000 Da. The isoelectric points are about 6 in both cases. Nuclear receptor can be extracted by 0.4M KCl. The binding can be completely restored if cytosol from the same source is incubated together with nuclei and [3H]20-hydroxyecdysone, but not if denatured cytosol or bovine serum albumin is used.

Animals↗
PubMed1981

Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus.

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.

Animals↗
PubMed1979

An in vitro culture system for crayfish organs.

Organs of crayfishes--mainly integument of Astacus leptodactylus--have been kept in an in vitro culture of several days. The medium consists of 2 parts of van Harreveld saline and one part of horse serum, supplemented with antibiotics for long term cultures. This medium is very similar to the Astacus hemolymph in its ionic content, the pH, the osmolarity, the protein concentration and the cholesterol content. The glucose concentration is somewhat higher and there are differences in the nitrogen excretory end products. Integument of Astacus is able to synthesize protein, RNA and DNA in vitro for several days. Ecdysone is converted to 20-OH-ecdysone by the integument under in vitro conditions.

Animals↗
PubMed1978

Immunohistochemical localisation of ecdysteroids in the follicular epithelium of locust oocytes.

Frozen sections of growing terminal follicles of the locust ovary were incubated with an ecdysteroid-specific rabbit antibody and the bound antibody visualised by the use of FITC-labelled goat-anti-rabbit antiserum. A bright fluorescence was seen in the cytoplasm of the follicle cells in terminal follicles with a length between 4.0 and 6.0 mm with a maximum intensity at 5.5 mm, indicating the presence of ecdysteroids in these cells in this particular developmental stage.

Animals↗
PubMed1978

A radioimmunoassay for arthropod moulting hormones, introducing a novel method of immunogen coupling.

Inokosteron-26-oic acid was coupled to thyroglobulin in aqueous pyridine by a water-soluble carbodiimide. After exhaustive dialysis and gel filtration on Sephadex G-25 in the presence of sodium dodecylsulfate, a coupling ratio of 164 haptens per molecule of thyroglobulin was determined. In all three animals injected with the conjugate, ecdysone-binding antibodies were detected. After one booster injection the antiserum could be diluted 1 : 5000 (1 : 4000, or 1 : 2000) in order to get a 50% binding of [3H]ecdysone. The dissociation constant was calculated as 5.8 X 10(-10) MOL/L. The antiserum has a greater affinity for ecdysone and 22-isoecdysone than for all other ecdysteroids and steroids tested.

Animals↗
PubMed1977

Enzymatic and chemical synthesis of 3-dehydroecdysterone, a metabolite of the moulting hormone of insects.

3-Dehydroecdysterone, a less polar metabolite of ecdysterone, was synthesized in vitro using either an enzyme (edcysone oxidase, EC 1.1.3.16) or the chemical catalyst platinum. 3-Dehydroecdysterone was purified and characterized by its chromatographic behaviour, its staining properties and by UV, IR, NMR and mass spectroscopy. The compound is 1/15 as active as ecdysone in the Calliphora moulting hormone bioassay. Its biological relevance is discussed.

Alcohol Oxidoreductases↗
PubMed1976

Derivatives of the insect moulting hormone for affinity chromatography, and their biological activities.

Several derivatives of the arthropod moulting hormone have been synthesized which were coupled to AH Sepharose 4B yielding about 2 mumole ligand per g wet gel. As an indication of the suitability of the ligands for biological work the puff inducing capacity of their methyl esters was tested. The methyl ester of inokosterone-C-26-carboxylic acid possesses the highest biological activity; lower activities were obtained with the esters of ecdysterone-C-6-CM-oxime and ecdysterone hemisuccinates. Therefore, inokosterone-C-26-carbocylic acid should be a useful ligand for affinity chromatography of ecdysone recptors from insect tissues.

Cholestenes↗
PubMedDate not supplied

Action of brassinosteroids on the epithelial cell line from Chironomus tentans.

The two brassinosteroids, 22 S,23 S-homobrassinolide and 22 S,23 S-homocastasterone are weak competitors of the binding of [3H]ponasterone A to the intracellular ecdysteroid receptor from the epithelial cell line from Chironomus tentans. The relative affinities to the ecdysteroid receptor are 0.001 for both brassinosteroids as compared to 20-OH-ecdysone and 0.1 in comparison to ecdysone. Both substances exert morphological effects similar to those observed with 20-OH-ecdysone. Like moulting hormones both brassinosteroids inhibit chitin synthesis. However, these effects were observed only at rather high concentrations (10(-5) to 10(-4) M) which were cytotoxic for 22 S,23 S-homobrassinolide.

Acetylcholinesterase↗