Effects of 17β-estradiol on the distribution and activity of some oxydative enzymes of uterine and cervical epithelium in neonatal mice

Summary The following enzymes were studied histochemically in uterine and cervical epithelium from neonatal mice treated with 17-estradiol for the first four days after birth: NADH-, NADPH-, succinate-, -glycerophosphate-, lactate-, glucose-6-phosphate-, and 17-OH-steroid dehydrogenases.It was demonstrated that estradiol administration had a marked influence on distribution and activity of several of the enzymes compared with the control animals. In cervix there was an increase of activity for most of the enzymes, especially in the apical parts of the epithelium cells. The uterine epithelium was also estradiol sensitive as regards most enzymes, and in the case of glucose-6-phosphate dehydrogenase there was a dramatic enhancement of reaction in the uterus of the experimental animals. The differences obtained between cervical and uterine epithelium are described.17-OH-steroid dehydrogenase could not be detected histochemically in the present material.Supported by the Norwegian Research Council for Science and the Humanities.     

A histochemical study of the oxidation of 17β-estradiol in human term placenta at different incubation conditions

Summary The present study deals with the histochemical demonstration of 17-estradiol dehydrogenase in human term placenta using the polyvinyl alcohol method to reduce diffusion artefacts. Incubations took place with both NAD⁺ and NADP⁺ as coenzymes and at different pH values of the incubation medium. The NAD⁺ linked enzyme reaction showed a greater activity than the NADP⁺ linked, both in the trophoblast as well as in connective tissue. There were differences in staining intensity at the different pH values, and strongest reaction was observed using glycine-NaOH buffer pH 10 in the incubation medium. Owing to a non-enzymatically reduction of nitro blue tetrazolium by reduced NAD⁺, the demonstration of 17-estradiol dehydrogenase is independent of diaphorase at this high pH. The findings are discussed in relation to data about nothing dehydrogenase and biochemically determined pH optima for the enzymatic reactions dealt with in this work.The following Abbreviations are used in this Article NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PVA polyvinyl alcohol - tris tris (hydroxymethyl)-aminomethane - 17-OH-SDH 17-OH-steroid-dehydrogenase Supported by The Norwegian Research Council for Science and the Humanities. Skilful assistance of Mrs. E. Alvestad, Mrs. Aa. Flatnes and Mrs. F. Sørensen is greatfully acknowledged.     

<Emphasis Type="Italic">kitb</Emphasis>, a second zebrafish ortholog of mouse <Emphasis Type="Italic">Kit</Emphasis>

The large numbers of duplicated pairs of genes in zebrafish compared to their mammalian counterparts has lead to the notion that expression of zebrafish co-orthologous pairs in some cases can together describe the expression of their mammalian counterpart. Here, we explore this notion by identification and analysis of a second zebrafish ortholog of the mammalian Kit receptor tyrosine kinase (kitb). We show that in embryos, kitb is expressed in a non-overlapping pattern to that of kita, in the anterior ventral mesoderm, Rohon-beardRohon–Beard neurons, the otic vesicle, and trigeminal ganglia. The expression pattern of kita and kitb in zebrafish together approximates that of Kit in mouse, with the exception that neither zebrafish kit gene is expressed in primordial germ cells, a site of kit expression in the mouse embryo. In addition, zebrafish kita is expressed in a site of zebrafish primitive hematopoiesis but not required for blood development, and we fail to detect kitb expression in sites of zebrafish hematopoiesis. Thus, the expression and function of zebrafish kit genes cannot be described as a simple partition of the expression and function of mouse Kit. We discuss the possibility that these unaccounted for expression domains and functions are derived from more ancestral gene duplications and partitioning instead of the relatively recent teleost teleost-specific duplication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.     

The effect of polyvinyl alcohol and polyvinyl pyrrolidone on diffusion artifacts in lactate dehydrogenase histochemistry

Summary The effect of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), alone and in combination, on diffusion artifacts in histochemical incubations has been investigated using LDH as model enzyme. By measuring the amount of formazan in the medium at the end of the incubation it has been shown that both substances, but especially PVA, are effective in limiting diffusion. The significance of this is discussed in general as well as in relation to other procedures used to reduce diffusion artifacts.The Following Abbreviations are used in the Article NAD -Nicotinamide Adenine Dinucleotide - NADH -Nicotinamide Adenine Dinucleotide, Reduced form - PVA Polyvinyl alcohol - PVP Polyvinyl pyrrolidone - PMS Phenazine methosulfate - tris tris (hydroxymethyl)-aminomethan - Nitro-BT Nitro Blue Tetrazolium - LDH Lactic dehydrogenase     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Observations of terrestrial locomotion in wild Polypterus senegalus from Lake Albert,Uganda

Polypterids, the most basal actinopterygians, are a group of fish long-considered living fossils and holding a key position for understanding fish and tetrapod evolution. Knowledge of the natural history of Polypterus is limited, their having been studied in little detail since the early 1900s. The locomotory habits of wild Polypterus senegalus from Lake Albert, Uganda, were investigated in 2014. High-speed videography demonstrated the capability of large Polypterus to move overland successfully. Contrary to previous evidence, field observations found that terrestrial locomotion in Polypterus is not inherently restricted by body size. Evidence that Polypterus exhibit this behaviour as part of their natural life history can be found in the existence of environmental challenges and the presence of adaptations for amphibious life.