Regulatory effects of growth hormone, glucocorticoids, and thyroid hormone on the estrogen receptor level in the rat liver.

The multihormonal regulation of the estrogen receptor in the liver of female rats was studied under in vivo conditions. The steroid receptor level was assayed by hormone binding and specific mRNA analyzed by solution hybridization using a 35S-labeled RNA probe complementary to the ligand-binding domain of the estrogen receptor gene. Serum growth hormone levels were measured and correlated to the effects of glucocorticoid and thyroid hormone administration on the estrogen receptor expression. In animals subjected to adrenalectomy plus thyroidectomy, the estrogen receptor concentration was reduced from 59 fmol/mg cytosol protein to 10 fmol/mg protein (i.e., with 87% relative to control animals). Adrenalectomy or thyroidectomy alone caused a decrease with 14% and 66%, respectively. Substitution with 10 micrograms betamethasone and 1 microgram triiodothyronine daily for 9 days completely restored the receptor content to control levels. Substitution with either hormone alone increased, but only partially restored receptor levels. The effect of betamethasone alone was dose dependent from 10 micrograms/d to 100 micrograms/d. This dose dependence was not seen when the animal simultaneously received 1 microgram of triiodothyronine. Superphysiologic doses of triiodothyronine did not raise estrogen receptor levels above those seen in animals treated with physiologic doses. High doses of triiodothyronine (greater than 20 micrograms/d) decreased serum growth hormone levels. The estrogen receptor mRNA levels in livers from hypophysectomized animals were increased after treatment with growth hormone (2.5-fold), thyroid hormone (two-fold), and glucocorticoids (1.5-fold). The results obtained indicate a very complex regulation of liver estrogen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic hydrolysis of lignocellulosic materials: II. Experimental investigations of theoretical hydrolysis--process models for an increased enzyme recovery

Stream pretreatment of wheat straw solubilized most of the xylan present. Xylose and other sugars were recovered by washing the substrate with water but only a minor part (34%) was monomeric. Treatment of this solutions with celulases and hemicellulases improved the yield of monomeric sugars to 69%, the main product being xylose. Some xylose was also obtained during enzymatic hydrolysis of the solid substrate although the pretreatment step contributed 64% (mean value) of total xylose formed. A reference model, No. 1, and two other models, Nos. 2 and 4, described in the first part of this article series (this issue) have been studied experimentally and results confirm the theoretical conclusions. An uninterrupted hydrolysis over a given time period leads to a lower degree of saccharification than when hydrolysate is withdrawn several times. Saccharification is also favored if the residue is removed at a late stage, i.e., at the end of the 24 h hydrolysis cycle. Extended recirculation of the enzymes during a 4 x 24-h experimental period gave the following average yields of saccharification on a 24-h basis: 65% (Reference), 73% (Model 2), and 79% (Model 4). It is concluded that enzyme recovery with model 4 is 70% or more, while the Reference and Model 2 attain a lower level of recovery. The design of an improved hydrolysis model is also discussed.     

Localisation and activation of the neurokinin 1 receptor in the enteric nervous system of the mouse distal colon

The substance P neurokinin 1 receptor (NK₁R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK₁R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK₁R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK₁R immunoreactivity (NK₁R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK₁R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK₁R-IR was expressed by a subpopulation of myenteric and submucosal neurons; it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK₁R-immunoreactive neurons were positive for nitric oxide synthase. NK₁R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK₁R from the cell surface into endosome-like structures. Myenteric NK₁R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK₁R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK₁R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK₁R in regulating intestinal motility and secretion.     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light-dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights-off and declined around lights-on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights-on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights-on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights-on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase-shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

Properties of the permeability transition in VDAC1(-/-) mitochondria

Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca2+ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a approximately 32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812-49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1(-/-) mice. The basic properties of the PTP in VDAC1(-/-) mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1(-/-) mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.     

Fleshy fruits – origins, niche shifts, and diversification

We examined shifts in fruit type, fleshy vs non-fleshy, in relation to habitat-related niche shifts, species richness, and historical distribution, in 50 phylogenetically independent plant lineages. Each lineage consisted of a sister-group pair of fleshy vs non-fleshy taxa and their outgroup. Niche shifts were assessed based on plant community characteristics. Two niche dimensions assumed to reflect community dynamics were derived: spatial predictability of disturbances and canopy closure. Phylogenetically independent origins of fleshy fruit types (1) were correlated with changes to habitats characterized by more shaded and spatially more unpredictable disturbances, (2) had an opposite effect on species richness in woody and herbaceous clades, enhancing species richness in woody clades, and (3) were continuously distributed over a period covering the last 70 million years. These results support the hypothesis that fleshy fruit evolution is driven by vegetation dynamics, and suggest that the strength of frugivore mediated selection on fleshy fruits increases when recruitment sites are spatially unpredictable and/or characterized by low light conditions.     

Evaluation of Bacillus anthracis thymidine kinase as a potential target for the development of antibacterial nucleoside analogs

Bacillus anthracis, which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) (Ba-TK) was cloned and expressed in E. coli, and the product was purified and characterized regarding its substrate specificity. Ba-TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba-TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low K(m) value (0.6 microM) and a V(max) of 3.3 micromol dTMP mg(-1) min(-1), but deoxyuridine (K(m)=4.2 microM, V(max)=4.1 micromol dUMP mg(-1) min(-1)) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3'- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro. These results may be used to guide future development of nucleoside analogs against B. anthracis.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

Social inequalities and mortality in europe - results from a large multi-national cohort

Background

Socio-economic inequalities in mortality are observed at the country level in both North America and Europe. The purpose of this work is to investigate the contribution of specific risk factors to social inequalities in cause-specific mortality using a large multi-country cohort of Europeans.

Methods

A total of 3,456,689 person/years follow-up of the European Prospective Investigation into Cancer and Nutrition (EPIC) was analysed. Educational level of subjects coming from 9 European countries was recorded as proxy for socio-economic status (SES). Cox proportional hazard model''s with a step-wise inclusion of explanatory variables were used to explore the association between SES and mortality; a Relative Index of Inequality (RII) was calculated as measure of relative inequality.

Results

Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest (HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78). The risk reduction was attenuated by 7% in men and 3% in women by the introduction of smoking and to a lesser extent (2% in men and 3% in women) by introducing body mass index and additional explanatory variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women). Social inequalities were highly statistically significant for all causes of death examined in men. In women, social inequalities were less strong, but statistically significant for all causes of death except for cancer-related mortality and injuries.

Discussion

In this European study, substantial social inequalities in mortality among European men and women which cannot be fully explained away by accounting for known common risk factors for chronic diseases are reported.