How We Raise Clean, Healthy \u3ci\u3eAmoeba proteus\u3c/i\u3e

We so frequently use Amoeba proteus for observation and experimentation in our classes in Cell Structure and Function, and Physiological Protozoology at the University of Kansas, that it has been necessary to avoid buying the many needed cultures from any of the major biological suppliers. Consequently, we have had to devise a reasonably reliable method of our own to culture that amoeba

How We Raise \u3ci\u3eParamecium\u3c/i\u3e, Easily, in Large Numbers

We use more Paramecium spp. in our Physiological Protozoology course at the University of Kansas than any other cells, and use them in great numbers each week, so that we wish to avoid the cost of buying them commercially. Therefore, we raise our own cultures. For over twenty years the senior author has used the easy method described here to raise large numbers of Paramecium aurelia, P. cauclatum, or P. multimicronucleatum, with consistent success

In silico prioritization of endocrine active substances (EAS) and their in vitro validation

In silico molecular docking can be a cheap and fast strategy to estimate the binding free energies, and consequently the dissociation constants, for a set of compounds with respect to their putative targets. Interesting targets for EAS are the ligand binding domains (LBD) of the human nuclear receptors for the sex hormones, i.e. the estrogen, androgen, progesterone, and (gluco)corticoid receptor. The Horizon 2020 project EuroMix (http://euromixproject.eu) aims to establish and disseminate new, efficient and validated strategies for the risk assessment of mixtures, while limiting the use of test animals. The present presentation deals with a part of EuroMix that is intended to set up a testing approach for mixtures of endocrine disrupting chemicals, focusing on estrogenic and anti-androgenic effects. For that purpose, a combined Adverse Outcome Pathway (AOP) was constructed, including Molecular Initiating Events, Key Events, and Adverse Outcome (reproductive dysfunction). Using this combined AOP as framework, cognate in silico and in vitro tools as well as the in vivo confirmation studies were selected, i.e. in silico: h-ER and h-AR docking; in vitro: cell-based ER and AR transcriptional activation bioassays and the H295R steroidogenesis assay; and in vivo: the Fish Sexual Development Test (FSDT, OECD Test No. 234) and a rat study, examining in (male) offspring a number of parameters, such as anogenital distance, cryptorchidism, and nipple retention. Reference chemicals were selected and in silico and in vitro testing was started, showing that when testing single compounds, there is a very good correlation between the in silico determined binding energies and the in vitro measured hormonal activities

Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T

Proton gradient formation in early endosomes from proximal tubules

AbstractHeavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (ΔpH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (Km = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H+-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl−-channel, Na+/H+-exchanger) present in early endosomas isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient