Mutagenicity testing of dichloromethane in short-term mammalian test systems

Several short-term mammalian test systems were used for mutagenicity testing of the organic solvent dichloromethane. The compound was negative in the forward mutation test on the HGPRT locus in Chinese hamster cells and the unscheduled DNA synthesis test in both human and hamster cells. In the test on DNA synthesis inhibition, dichloromethane caused an aspecific inhibition in both human and hamster cells, but in this test the effect did not indicate a DNA-damaging action. A weak positive effect was found in the test on sister-chromatid exchanges in hamster cells.     

What life cycle graphs can tell about the evolution of life histories

We analyze long-term evolutionary dynamics in a large class of life history models. The model family is characterized by discrete-time population dynamics and a finite number of individual states such that the life cycle can be described in terms of a population projection matrix. We allow an arbitrary number of demographic parameters to be subject to density-dependent population regulation and two or more demographic parameters to be subject to evolutionary change. Our aim is to identify structural features of life cycles and modes of population regulation that correspond to specific evolutionary dynamics. Our derivations are based on a fitness proxy that is an algebraically simple function of loops within the life cycle. This allows us to phrase the results in terms of properties of such loops which are readily interpreted biologically. The following results could be obtained. First, we give sufficient conditions for the existence of optimisation principles in models with an arbitrary number of evolving traits. These models are then classified with respect to their appropriate optimisation principle. Second, under the assumption of just two evolving traits we identify structural features of the life cycle that determine whether equilibria of the monomorphic adaptive dynamics (evolutionarily singular points) correspond to fitness minima or maxima. Third, for one class of frequency-dependent models, where optimisation is not possible, we present sufficient conditions that allow classifying singular points in terms of the curvature of the trade-off curve. Throughout the article we illustrate the utility of our framework with a variety of examples.     

Demonstration of choleragen-dependent ADP-ribosylation in whole cells and correlation with the activation of adenylate cyclase

3T3C₂ mouse fibroblasts rendered permeable to (α^?32P)NAD⁺ show cholera toxin-dependent labeling of a 45,000 m.w. protein and of a doublet of polypeptides around 52,000 m.w. These same bands are ADP-ribosylated in broken cells. Membranes prepared from pigeon erythrocytes pretreated with choleragen show a decrease in subsequent cholera toxin-specific ADP-ribosylation of a 43,000 m.w. polypeptide. Both whole cell and broken cell adenylate cyclase activation and toxin-specific ADP-ribosylation are reversed specifically by low pH and high concentrations of toxin and nicotinamide in all systems. Thus ADP-ribosylation appears to be relevant to the molecular action of choleragen in whole cells as well as in broken cells.     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Explaining density-dependent regulation in earthworm populations using life-history analysis

At present there is little knowledge about how density regulates population growth rate and to what extent this is determined by life-history patterns. We compared density dependent population consequences in the Nicholsonian sense based on experimental observations and life-history modeling for the earthworms Lumbricus terrestris and Eisenia fetida . Both species differ in their life-histories, L. terrestris being a relatively long-lived species with slow reproduction and occurring at low densities compared to E. fetida which has a more opportunistic strategy with a high reproductive output. E. fetida is able to colonise new habitats rapidly and may occur at relatively high population densities. Density dependency of population growth rate was estimated by incorporating density dependent effects on reproduction and growth using a modified Euler equation. The results point out that E. fetida was not as strongly impacted by density as compared to L. terrestris . Population growth rate in E. fetida was hardly affected at low and moderate density, being reduced only at high level, this compares to L. terrestris where even relatively small density effects resulted in a strong negative effect on population growth rate. Our findings indicate that density-dependent regulation in earthworms can be quantified using life-history analysis. The outcomes are in agreement with empirical field observations for populations (i.e. L. terrestris occurs ar low density, E. fetida at high density). Consideration of the potential importance of Nicholsonian density dependence for field populations of these two species in light of their known biology however produces counterintuitive conclusions. In E. fetida , although density tolerant, rapid population growth may mean this species may be subject to density dependeny regulation. In L. terrestris , although density sensitive, complex behavioural ecology (surface activity, territoriality) may limit of feedback influence on population size.     

5 alpha-androstane-3 beta,17 beta-diol hydroxylating enzymes in stroma and epithelium of human benign prostatic hyperplasia (BPH)

As enzymatic hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) may be a factor in controlling the 5 alpha-dihydrotestosterone (DHT) content in the prostate, we were interested in activity and distribution of these enzymes in epithelium and stroma of human benign prostatic hyperplasia (BPH). The enzyme activities were measured after mechanical separation of BPH tissue from 15 patients of various ages into stroma and epithelium, and optimization of the in vitro transformation of 3 beta-diol to hydroxylated products, which were analyzed by HPLC. The main results were: (1) 3 beta-diol was hydroxylated at C-7 alpha, C-7 beta, C-6 alpha, and C-6 beta. (2) The mean Michaelis constant Km (nM +/- SEM) for hydroxylation at C-7 alpha(beta) (168 +/- 21) was significantly lower than at C-6 alpha(beta) (601 +/- 43) without differences between stroma and epithelium. (3) Hydroxylation at alpha position dominated significantly over that at beta. (4) The mean maximal metabolic rate Vmax (pmol . mg protein-1 . h-1) of hydroxylation at C-6 alpha was about 7-fold lower in stroma (3.4 +/- 0.2) than in epithelium (23.8 +/- 4.1), concerning the other hydroxylations, Vmax was about 1.6-fold lower in stroma. (5) With increasing age of the patients there was a significant decrease of the 3 beta-diol hydroxylation in stroma and epithelium. It is discussed that the significantly lower activity of 3 beta-diol hydroxylation in stroma compared to epithelium and the decrease of activity with increasing age might potentiate the DHT accumulation in stroma of BPH.     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Mesotocin and vasotocin in the brain of the lizard Gekko gecko

Summary The distribution of mesotocin and vasotocin was studied in the brain of the lizard Gekko gecko with antisera specific for either peptide. Both mesotocinergic and vasotocinergic perikarya are found in the paraventricular and supraoptic nuclei of the hypothalamus, whereas vasotocinergic neurons are exclusively present in the bed nucleus of the stria terminalis and in a cell group of the rhombencephalon. The distributional pattern of the mesotocinergic fibers corresponds closely to that of the vasotocinergic fibers. However, throughout the entire brain the mesotocinergic innervation is less dense than the vasotocinergic innervation. No sex differences are present in the mesotocinergic fiber system.Abbreviations acc nucleus accumbens - bst bed nucleus of the stria terminalis - bv blood vessel - dB diagonal band of Broca - dc dorsal cortex - dth dorsolateral thalamic nucleus - lc lateral cortex - me median eminence - oc optic chiasma - ot optic tract - pag periaqueductal grey - pvn paraventricular nucleus - rc rhombencephalic cell group - sep septum - son supraoptic nucleus - tect mesencephalic tectum - vth ventrolateral thalamus     

Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.