Testosterone Restoration by Enclomiphene Citrate in Men with Secondary Hypogonadism: a pharmacodynamic and Pharmacokinetic study.

OBJECTIVES: To determine the pharmacodynamic (PD) profile of serum total testosterone levels (TT) and luteinizing hormone (LH) in men with secondary hypogonadism following initial and chronic daily oral doses of enclomiphene citrate in comparison to transdermal testosterone. To determine the effects of daily oral doses of enclomiphene citrate (Androxal®) in comparison to transdermal testosterone on other hormones and markers in men with secondary hypogonadism. PATIENTS AND METHODS: This was a randomized, single blind, two-center phase II study to evaluate three different doses of enclomiphene citrate (6.25mg, 12.5mg and 25 mg Androxal®), versus AndroGel®, a transdermal testosterone, on 24-hour LH and TT in otherwise normal healthy men with secondary hypogonadism. Forty-eight men were enrolled in the trial (ITT Population), but 4 men had T levels >350 ng/dL at baseline. Forty-four men completed the study per protocol (PP population). All subjects enrolled in this trial had serum TT in the low range (<350 ng/dL) and had low to normal LH (<12 IU/L) on at least two occasions. TT and LH levels were assessed each hour for 24 hours to examine the effects at each of three treatment doses of enclomiphene versus a standard dose (5 grams) of transdermal testosterone (AndroGel). In the initial profile TT and LH were determined in a naïve population following a single initial oral or transdermal treatment (Day 1). This was contrasted to that seen after six weeks of continuous daily oral or transdermal treatment (Day 42). The pharmacokinetics of enclomiphene was performed in a select subpopulation. Serum samples were obtained over the course of the study to determine levels of various hormones and lipids. RESULTS: After six weeks of continuous use, the mean ± SD concentration of TT at Day 42 C0hrTT, was 604 ± 160 ng/dL for men taking the highest of dose of enclomiphene citrate (enclomiphene, 25 mg daily) and 500 ± 278 ng in those men treated with transdermal testosterone. These values were higher than Day 1 values but not different from each other (p = 0.23, T-test). All three doses of enclomiphene increased C0hrTT, CavgTT, CmaxTT, CminTT and CrangeTT. Transdermal testosterone also raised TT, albeit with more variability, and with suppressed LH levels. The patterns of TT over 24 hour period following six weeks of dosing could be fit to a non-linear function with morning elevations, mid-day troughs, and rising night-time levels. Enclomiphene and transdermal testosterone increased levels of TT within two weeks, but they had opposite effects on FSH and LH Treatment with enclomiphene did not significantly affect levels of TSH, ACTH, cortisol, lipids, or bone markers. Both transdermal testosterone and enclomiphene citrate decreased IGF-1 levels (p<0.05) but suppression was greater in the enclomiphene citrate groups. CONCLUSIONS: Enclomiphene citrate increased serum LH and TT; however, there was not a temporal association between the peak drug levels and the Cmax levels LH or TT. Enclomiphene citrate consistently increased serum TT into the normal range and increased LH and FSH above the normal range. The effects on LH and TT persisted for at least one week after stopping treatment.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

Evaluation of a Single Dose of Ferric Carboxymaltose in Fatigued, Iron-Deficient Women - PREFER a Randomized, Placebo-Controlled Study

BACKGROUND: Unexplained fatigue is often left untreated or treated with antidepressants. This randomized, placebo-controlled, single-blinded study evaluated the efficacy and tolerability of single-dose intravenous ferric carboxymaltose (FCM) in iron-deficient, premenopausal women with symptomatic, unexplained fatigue. METHODS: Fatigued women (Piper Fatigue Scale [PFS] score ≥5) with iron deficiency (ferritin <50 µg/L and transferrin saturation <20%, or ferritin <15 µg/L) and normal or borderline hemoglobin (≥115 g/L) were enrolled in 21 sites in Austria, Germany, Sweden and Switzerland, blinded to the study drug and randomized (computer-generated randomization sequence) to a single FCM (1000 mg iron) or saline (placebo) infusion. Primary endpoint was the proportion of patients with reduced fatigue (≥1 point decrease in PFS score from baseline to Day 56). RESULTS: The full analysis included 290 women (FCM 144, placebo 146). Fatigue was reduced in 65.3% (FCM) and 52.7% (placebo) of patients (OR 1.68, 95%CI 1.05-2.70; p = 0.03). A 50% reduction of PFS score was achieved in 33.3% FCM- vs. 16.4% placebo-treated patients (p<0.001). At Day 56, all FCM-treated patients had hemoglobin levels ≥120 g/L (vs. 87% at baseline); with placebo, the proportion decreased from 86% to 81%. Mental quality-of-life (SF-12) and the cognitive function scores improved better with FCM. 'Power of attention' improved better in FCM-treated patients with ferritin <15 µg/L. Treatment-emergent adverse events (placebo 114, FCM 209; most frequently headache, nasopharyngitis, pyrexia and nausea) were mainly mild or moderate. CONCLUSION: A single infusion of FCM improved fatigue, mental quality-of-life, cognitive function and erythropoiesis in iron-deficient women with normal or borderline hemoglobin. Although more side effects were reported compared to placebo, FCM can be an effective alternative in patients who cannot tolerate or use oral iron, the common treatment of iron deficiency. Overall, the results support the hypothesis that iron deficiency can affect women's health, and a normal iron status should be maintained independent of hemoglobin levels. TRIAL REGISTRATION: ClinicalTrials.gov NCT01110356.

In vitro compatibility of remifentanil hydrochloride and sufentanil citrate with selected drugs

: Objectives Physicochemical incompatibilities between intravenous drugs are a recurrent problem in intensive care units. The present study was aimed at investigating the physical compatibility of remifentanil and sufentanil with other drugs (insulin, midazolam, propofol, potassium chloride, magnesium sulfate, furosemide, heparin, monobasic potassium phosphate) that are frequently administered together intravenously. In addition, the physicochemical compatibility of three common associations of drugs was evaluated in glass tube tests and during dynamic simulated Y site administrations (remifentanil-insulin-midazolam; remifentanil-insulin-propofol; sufentanil-insulin-midazolam). Methods Physical compatibility was verified by visual inspection of the various mixtures (two, three or four drugs) in glass tubes and by pH determination of the mixtures collected during simulated Y site administrations. Solutions were considered as compatible in the absence of any visual change in the solution and of any significant variation in pH value. In addition, chemical stability was checked during in vitro dynamic simulations. The solutions were prepared in 50 ml syringes, placed on syringe pumps and connected to a Swan-Ganz catheter; the liquid collected at the tip was assayed by high performance liquid chromatography. Results In the visual examinations, only the associations of remifentanil and furosemide were incompatible. The three assayed associations were compatible in the tested proportion range over 24 h. Conclusions Remifentanil was physically compatible with the tested drugs, except for furosemide (Lasix; Sanofi-Aventis, 250 mg/25 ml) and physicochemically compatible with insulin and midazolam and insulin and propofol. Sufentanil was physically compatible with all tested drugs and physicochemically compatible with insulin and midazolam

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes.

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.

ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

Quelles applications raisonnables pour l'anticoagulation régionale au citrate en épuration extrarénale [What reasonable applications for regional citrate anticoagulation in renal replacement therapy?].

Regional citrate anticoagulation of the extracorporeal circuits (CRA) experienced considerable growth over the past decade. This development is partly explained by the significant progresses made in the field of bioengineering. These allow a secure administration of citrate, while an increasing availability of ionized calcium measurement at the bedside allows reactivity in monitoring the treatment. An increasing severity of the medical condition of patients requiring blood purification treatment gives more contrast to the profile of patient who may benefit from a CRA. If some methods of renal replacement therapy are well suited to this mode of anticoagulation, others are, to date, only at the stage of development and are applied under close medical supervision.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

High-resolution magnetic resonance imaging quantitatively detects individual pancreatic islets.

OBJECTIVE-We studied whether manganese-enhanced high-field magnetic resonance (MR) imaging (MEHFMRI) could quantitatively detect individual islets in situ and in vivo and evaluate changes in a model of experimental diabetes.RESEARCH DESIGN AND METHODS-Whole pancreata from untreated (n = 3), MnCl(2) and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl(2) and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Testa scanner and correlated with the corresponding (immuno)histological sections.RESULTS-In ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of beta-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individual islets in living, anesthetized mice.CONCLUSIONS-Data show that MEHFMRI quantitatively visualizes individual islets in the intact mouse pancreas, both ex vivo and in vivo. Diabetes 60:2853-2860, 2011

Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa.

The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pchEF, encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE (156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensation/cyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchE'-'lacZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led to Fur (ferric uptake regulator)-dependent repression of the pchE'-'lacZ fusion. A translational pchD'-'lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

Detection of fingermarks by colloidal gold (MMD/SMD): beyond the pH 3 limit

This work is part of a continuing goal to improve the multimetal deposition technique (MMD), as well as the single-metal deposition (SMD), to make them more robust, more user-friendly, and less labour-intensive. Indeed, two major limitations of the MMD/SMD were identified: (1) the synthesis of colloidal gold, which is quite labour-intensive, and (2) the sharp decrease in efficiency observed when the pH of the working solution is increased above pH 3. About the synthesis protocol, it has been simplified so that there is no more need to monitor the temperature during the synthesis. The efficiency has also been improved by adding aspartic acid, conjointly with sodium citrate, during the synthesis of colloidal gold. This extends the range of pH for which it is possible to detect fingermarks in the frame of the MMD/SMD. The operational range is now extended from 2 to 6.7, compared to 2-3 for the previous formulations. The increased robustness of the working solution may improve the ability of the technique to process substrates that tend to increase the pH of the solution after their immersion.

Arrested kinetic Li isotope fractionation at the margin of the llimaussaq complex, South Greenland: Evidence for open-system processes during final cooling of peralkaline igneous rocks

Li contents [Li] and isotopic composition (delta Li-7) of mafic minerals (mainly amphibole and clinopyroxene) from the alkaline to peralkaline Ilimaussaq plutonic complex, South Greenland, track the behavior of Li and its isotopes during magmatic differentiation and final cooling of an alkaline igneous system. [Li] in amphibole increase from < 10 ppm in Caamphiboles of the least differentiated unit to >3000 ppm in Na-amphiboles of the highly evolved units. In contrast, [Li] in clinopyroxene are comparatively low (<85 ppm) and do not vary systematically with differentiation. The distribution of Li between amphibole and pyroxene is controlled by the major element composition of the minerals (Ca-rich and Na-rich, respectively) and changes in oxygen fugacity (due to Li incorporation via coupled substitution with ferric iron) during magmatic differentiation. delta(7) Li values of all minerals span a wide range from + 17 to - 8 parts per thousand, with the different intrusive units of the complex having distinct Li isotopic systematics. Amphiboles, which dominate the Li budget of whole-rocks from the inner part of the complex, have constant delta Li-7 of + 1.8 +/- 2.2 parts per thousand (2 sigma, n = 15). This value reflects a homogeneous melt reservoir and is consistent with their mantle derivation, in agreement with published O and Nd isotopic data. Clinopyroxenes of these samples are consistently lighter, with Delta Li-7(amph-cpx). as large as 8 parts per thousand and are thus not in Li isotope equilibrium. These low values probably reflect late-stage diffusion of Li into clinopyroxene during final cooling of the rocks, thus enriching the clinopyroxene in 6 Li. At the margin of the complex delta(7) Li in the syenites increases systematically, from +2 to high values of + 14 parts per thousand. This, coupled with the observed Li isotope systematics of the granitic country rocks, reflects post-magmatic open-system processes occurring during final cooling of the intrusion. Although the shape and magnitude of the Li isotope and elemental profiles through syenite and country rock are suggestive of diffusion-driven isotope fractionation, they cannot be modeled by one-dimensional diffusive transport and point to circulation of a fluid having a high 67 Li value (possibly seawater) along the chilled contact. In all, this study demonstrates that Li isotopes can be used to identify complex fluid- and diffusion-governed processes taking place during the final cooling of such rocks. (c) 2007 Elsevier B.V All rights reserved.