The Effect of Maternal Aldosterone Levels on the Expression of 11B-HSD Isoenzymes in Normal and Hypertensive Rat Placentae

In this project, we developed a Western blotting procedure to semi-quantitate levels of 11β-HSD1 and 11β-HSD2 in whole cell extracts. Then, we applied this technique to analyze the effect of reduced maternal aldosterone levels on the expression of 11β-HSD1 and 11β-HSD2 isoenzymes in the placental tissue in both normal and hypertensive rats. These enzymes control levels of glucocorticoids which compete for aldosterone’s mineralocorticoid receptor. Overstimulation of this receptor results in hypertension. If aldosterone levels decrease, levels of the enzymes controlling active glucocorticoid concentrations might change to compensate for the lowered aldosterone levels. Decreased placental 11β-HSD2 expression could affect hypertension in the offspring. The Western blotting procedure was optimized for the detection of the two isoenzymes in their dimeric forms. The use of multiple protein levels on the blot was useful to obtain a more reliable semi-quantitation. Because of the presence of multiple bands, it was impossible to use an internal beta actin standard to normalize the data from one blot to another. This along with the low expression levels and uneven blot backgrounds made it difficult to obtain enough data on the expression of the two isoenzymes. Very limited data do not support differences in the placental expression of the two isoenzymes when maternal aldosterone levels are lowered

The Effect of Maternal Aldosterone Levels on the Expression of 11B-HSD Isoenzymes in Normal and Hypertensive Rat Placentae

The enzyme 11-betahydroxysteroid dehydrogenase 2 (1 lB-HSD 2) is important in hormone metabolism. It converts the active glucocorticoid to the inactive form so that it does not compete for the aldosterone receptor. Two isoforms of the enzyme are found in the placenta, but evidence supports a role for the HSD 2 enzyme in theprevention of fetal hypertension. Abnormalities in maternal hormones can affect hormonal expression in the offspring. This project is part of a larger hypertension study conducted by Dr. Knoblich. She is using cryotreatment of the adrenal gland to reduce aldosterone production in both normal and hypertensive female rats. We hope to study the effects of the reduced maternal aldosterone on placental expression of 11-B HSD 1 and HSD 2 in both normotensive rats (WKY) and hypertensive rats (SHR). The Western blotting technique will be used and protein levels will be quantitated by densitometry. Complementary studies on RNA expression are also being conducted in the lab. Initial experiments have focused on simple extraction methods followed by optimization of antibody levels using commercially available polyclonal antibodies and a sensitive chemiluminscent substrate. Consistent blots have been obtained; however, multiple bands are seen. Current experiments will test the effect of reducing agent in the extraction buffer. Once the technique is optimized, we can compare the effect of reduced maternal aldosterone levels on HSDl and 2 protein expression in the placentae. Along with the RNA data and the work of Dr. Knoblich\u27s research group, the results should contribute to the understanding of hormonal influences on hypertension

Phytochromes with noncovalently bound chromophores: The ability of apophytochromes to direct tetrapyrrole photoisomerization

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both cyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phyto chrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red- absorbing forms of phytochrome [P-r and P-fr forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P-r-P-fr, photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties P-r and P-fr forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light- induced physiological response

New X-ray sources detected among mild barium and S stars.

info:eu-repo/semantics/publishe

Two independent, light-sensing two-component systems in a filamentous cyanobacterium

Two ORFs, cphA and cphB , encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix , all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3' end and expressed in Escherichia coli . The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P-r and P-fr forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (lambda(max) = 685 and 735 nm for P-r and P-fr , respectively) is indicative for a noncovalent incorporation of the chromophore (lambda(max) of P- r , P-fr of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246-->Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P-fr formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis , showed differences in the kinetics of the P-fr formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore

Sequestered Chloroplasts In The Benthic Foraminifer Haynesina Germanica: Cellular Organization, Oxygen Fluxes And Potential Ecological Implications

Haynesina germanica is a coastal benthic foraminifer known to sequester chloroplasts from benthic pennate diatoms. This study investigates its cellular organization, as well as the oxygen consumption and production rates under dark and light conditions. The implications of the sequestered chloroplasts are subsequently discussed at an individual and ecological level. Living specimens collected in Arcachon Basin showed well-preserved chloroplasts in the cytoplasm in observations using transmission electron microscopy. Microelectrodes were used to measure oxygen consumption and production rates in incubated specimens in the laboratory. In the dark, oxygen consumption rates ranged from 662 +/- 12 to 1082 +/- 59 pmol O-2 ind(-1) d(-1). Under illuminated conditions (i.e., about 300 mu mol photon(-1) m(-2) s(-1)), we observed and quantified substantial photosynthetic activity, up to 199 pmol O-2 ind(-1) d(-1). It appears that H. germanica combines autotrophic and heterotrophic nutritional modes, thereby reflecting a mixotrophic strategy. In Arcachon Basin, H. germanica potentially uses chloroplasts acquired from microphytobenthos to carry out photosynthesis and produce oxygen and organic matter. Stored chloroplasts could constitute an additional food source during impoverished periods, providing substantial competitive advantage to this species. This putative behavior could also affect the biogeochemistry of the sediment at a microscale by creating oxygenated microniches around individual foraminifera

Rhinitis/Bronchopneumonia syndrome in Irish Wolfhounds.

This study describes the clinical, immunologic, genetic, and pathologic features of Irish Wolfhounds with rhinitis/bronchopneumonia syndrome. The dogs examined were from Belgium, The Netherlands, UK, Canada, Germany, and Switzerland. Signs included transient to persistent mucoid or mucopurulent rhinorrhea, cough, and respiratory dyspnea. Radiographic, rhinoscopic, and bronchoscopic findings were variable. Analysis of ciliary ultrastructure was performed in 5 affected dogs, but no characteristic primary ciliary defects (primary ciliary dyskinesia) were detected. Serum and bronchoalveolar lavage fluid (BALF) concentrations of IgA, IgG, and IgM were determined in some affected dogs and clinically normal Irish Wolfhounds. Serum IgA concentration was below the reference range in 5 of 8 affected dogs tested, whereas BALF IgA concentration was above the normal range in 2 affected adult dogs. The CD4 to CD8 lymphocyte subset ratio (CD4:CD8) in peripheral blood was tested in 3 affected dogs and was within the normal range. BALF CD4:CD8 was tested in 1 affected dog and was higher than the normal range. Decreased neutrophil phagocytosis was observed in 1 of the 4 dogs tested. Analysis of pedigrees of the Belgian, Canadian, German, and Swiss dogs revealed common ancestry, suggesting a heritable syndrome.Journal Articleinfo:eu-repo/semantics/publishe

Marine nitrogen cycling dynamics under altering redox conditions: Insights from deposition of sapropels S1 and the ambiguous S2 in the Eastern Mediterranean Sea

International audienceThe eastern Mediterranean Sea (EMS) sedimentary record is periodically interspersed with organic-rich 'sapropel' layers. Sapropels are characteristic of basin-wide anoxic events, triggered by precession-forced insolation maxima. Relatively subdued insolation maxima, however, are not always expressed as distinct sapropel events. The EMS sedimentary record is thus useful to investigate feedbacks between marine anoxia and the nitrogen (N) cycle and offers an analogue for modern deoxygenation and past oceanic anoxic events. To this end, we investigated a ~68 kyr sedimentary record from the EMS containing the well-established sapropel S1 (deposited in two phases: S1a [~10.5-8.5 ka BP] and S1b [~7.8-6.1 ka BP]) and sediments timed to the ambiguous S2 sapropel (~53 ka BP). We used lipid biomarkers of microorganisms to reconstruct key N-cycle components: (1) anaerobic ammonium oxidation (anammox) using ladderanes and a stereoisomer of bacteriohopanetetrol (BHTx), (2) dinitrogen gas (N 2) fixation using heterocyte glycolipids, and (3) nitrification by Thaumarchaeota using crenarchaeol. Additionally, benthic foraminifera and trace metals (U, Mo, Mn) were used to reconstruct redox conditions. During S1a, abundances of Thaumarchaeota increased, likely promoted by elevated high-nutrient freshwater discharge. At this time, a combination of phosphorus supply and intensified loss of bioavailable N via water column anammox, may have reinforced anoxia by favoring diatom-diazotroph associations. During S1b, anammox is equally intense. Yet, no positive feedback on N 2-fixation is observed, likely because diazotrophs were phosphorus limited. Instead, anammox may have provided negative feedback on anoxia by quenching primary production. Ladderanes suggest additional episodes of anammox between ~69 to 39 cal ka BP, corresponding to brief periods of water column deoxygenation. Anoxia likely occurred at the sediment-water interface in S2-timed sediments (53-51 cal ka BP). During these episodes, ladderanes co-occur with the later eluting BHT-34R stereoisomer. δ 13 C BHT-34R indicate an anammox source, potentially synthesized by marine sedimentary anammox bacteria. No corresponding increase in diatom-diazotroph associations is observed, likely due to the oligotrophic conditions and the limited effect of sedimentary anammox on N-availability in the euphotic zone. Our results highlight various modes of operation of the N-cycle at different degrees of deoxygenation, which depend amongst others on nutrient-availability and the niche-segregation of N-loss and N 2-fixating microorganisms