Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in diluted overnight urine. An approach to the study of luteal insufficiency

The determination of the concentrations of estrone-3-glucuronide, pregnanediol-3-glucuronide and luteinizing hormone has been performed in early morning urine samples of 14 normal menstruating women using a timed and measured volume urine collection procedure. In order to investigate the variability of the urinary hormonal concentrations due to day-to-day differences in diuresis, the absolute hormonal concentrations have been corrected either for the urinary creatinine excretion or for the volume of urine voided during the night. The results demonstrate that both correction factors are able to reduce substantially the coefficient of variation values in comparison to the absolute hormonal concentrations. The urinary test of ovarian function has been performed in 11 infertile women affected by luteal insufficiency using the same procedure, and the hormonal profiles showed some alterations in both estrone-3-glucuronide and pregnanediol-3-glucuronide concentrations in comparison to the hormonal profiles of the normal subjects. Such alterations were significant in the single subject when integrated values of the hormonal data in defined time intervals were investigated.     

Immunolocalization of inhibin α-subunit in the human testis

Summary The localization of inhibin -subunit in the human testis was studied at the light- and electron-microscope level with immunostaining techniques. Antibodies against specific fragments of porcine and human inhibin -subunits were utilized. At light microscopy, inhibin -subunit immunoreactivity was detected in Sertoli cells, spermatocytes and in some Leydig cells. At electron microscopy, gold labeling was found in the cisternae of the Golgi apparatus and in the endoplasmic reticulum of Sertoli and Leydig cells. Gold labeling for inhibin was also found in coated vesicles in the cytoplasm of Sertoli cells as well as in coated pits and coated vesicles in the cytoplasm of some spermatocytes. The results of the present study suggest that, in the human testis, inhibin is produced by Sertoli and Leydig cells and is taken up by spermatocytes, on which it might act in a paracrine manner.     

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2′-deoxy-N₆-methyl adenosine 3′,5′-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS 2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned.     

Integrated in vitro approaches to assess the bioaccessibility and bioavailability of silicon-biofortified leafy vegetables and preliminary effects on bone

Food industries are increasingly oriented toward new foods to improve nutritional status and/or to combat nutritional deficiency diseases. In this context, silicon biofortification could be an innovative tool for obtaining new foods with possible positive effects on bone mineralization. In this paper, an alternative and quick in vitro approach was applied in order to evaluate the potential health-promoting effects of five silicon-biofortified leafy vegetables (tatsoi, mizuna, purslane, Swiss chard and chicory) on bone mineralization compared with a commercial silicon supplement. The silicon bioaccessibility and bioavailability of the five leafy vegetables (biofortified or not) and of the supplement were assessed by applying a protocol consisting of in vitro gastrointestinal digestion coupled with a Caco-2 cell model. Silicon bioaccessibility ranged from 0.89 to 8.18 mg/L and bioavailability ranged from 111 to 206 μg/L of Si for both vegetables and supplement. Furthermore, the bioavailable fractions were tested on a human osteoblast cell model following the expression of type 1 collagen and alkaline phosphatase. The results obtained highlighted that the bioavailable fraction of biofortified purslane and Swiss chard improved the expression of both osteoblast markers compared with the supplement and other vegetables. These results underline the potentially beneficial effect of biofortified leafy vegetables and also indicate the usefulness of in vitro approaches for selecting the best vegetable with positive bone effects for further in vivo research.     

High levels of genetic diversity in populations of Iris aphylla L. (Iridaceae), an endangered species in Poland

We used RAPDs (Random Amplified Polymorphic DNAs) to investigate genetic diversity and its partition within and between three populations of Iris aphylla in Poland. Analysis of Molecular Variance (AMOVA) of 84 distinct RAPD multiband genotypes revealed higher variation within populations (77.2%) than genetic differentiation between them (22.8%, P  < 0.002). Values of genetic diversity indices ( H ) were similar in all three sites (0.21–0.24). The differentiation of the populations corresponded to low average gene flow ( Nm  = 0.81). Our results indicated that genetic diversity was independent of population size. We concluded that although sexual reproduction and gene flow between populations of I. aphylla were very limited, they preserved high levels of genetic diversity. Relatively large number of seeds, which migrated in the past to populations, as well as patterns of reproduction and life history of I. aphylla may explain this situation.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 65–72.     

Local expression of enzymatically active class I β-1, 3-glucanase enhances symptoms of TMV infection in tobacco

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.     

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA‐based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z‐nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate‐specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z‐specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome‐wide mapping reveals that PWWP2A binds selectively to H2A.Z‐containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C‐terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z‐specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.     

Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine

The relationships of the electrical to the mechanical responses of the canine trachealis muscle during stimulation of its cholinergic nerves or exposure to exogenous acetylcholine were recorded in the single or the double sucrose gap. At 27 degrees C, the responses to a train of stimuli consisted of a transient depolarization excitatory junction potential of 10-30 mV followed by fading oscillations and contractions. When stimulus parameters were varied in the single sucrose gap, contractions were more closely associated with the occurrence of and varied in duration with the oscillations rather than with the amplitude of the EJP. Acetylcholine superfused at a concentration of 10(-6) M for 30 s caused a prolonged depolarization of 10-20 mV, but a much larger contraction than could be elicited by nerve stimulation. None of the responses to acetylcholine was significantly affected by the Ca channel antagonists, nifedipine, nitrendipine, or verapamil in Ca channel blocking concentrations. When tissues were exposed to a Ca-free medium, the excitatory junction potentials and oscillations rapidly disappeared, but the electrical and mechanical responses to acetylcholine persisted and only gradually disappeared with repetitive exposures. Furthermore, in a medium with normal Ca2+ in the double sucrose gap, depolarization by 10-15 mV with an applied current caused no contraction, and repolarization to the normal membrane potential during acetylcholine-induced contraction caused no relaxation. Tetraethylammonium ion (20 mM) depolarized the membrane, increased membrane resistance, and enhanced the secondary oscillations and contractions after field stimulation. No other K(+)-channel blocker tested (Ba2+, apamin, 4-aminopyridine, glibenclamide, charybdotoxin) had the effect of prolonging secondary oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)