Pivaloylcodeine,a new codeine derivative,for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC₅₀ were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC₅₀ 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

Inter-simple sequence repeat markers and flow cytometry for the characterization of closely related Citrus limon germplasms

The genetic relationship among commercial cultivars of Citrus limon (lemon) was analysed by inter-simple sequence repeats (ISSR) and flow cytometry techniques. Two cultivars with a close germplasm were distinguished by screening 10 SSR primers and by measuring DNA content of prestained nuclei.     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

The source of ascitic fluid in experimental cirrhosis in the rat.

In order to disclose the source of ascitic fluid in liver cirrhosis, normal and cirrhotic rats were injected with fluorescein into the paracecal vein. The green fluorescence was then evaluated on the surface of the liver, the intestine and the peritoneum. Among healthy rats and in those with anascitic cirrhosis a very slight fluorescence was detected on the liver capsule whereas among rats with ascitic cirrhosis a distinct fluorescence was shown on the liver surface, the small intestine and the peritoneum. Therefore, the peritoneum is a source of ascitic fluid in cirrhosis of the rat.     

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Background  

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.     

Nitrate reductase activity,biomass, yield,and quality in cotton in response to nitrogen fertilization

In the production of cotton (Gossypium hirsutum L.), nitrogen fertilization is one of the most costly crop practices, but important to reach high yields. However, high nitrogen (N) content in plants does not always translate into a high fibre production. One way of assessing the efficiency of the N fertilizer is through the enzymatic activity of the nitrate reductase (NR). This is a key enzyme in N assimilation, whose activity is regulated by a number of endogenous and exogenous factors that determine yield. The aim of this study was to assess the effect of N fertilization on yield, fibre quality, biomass, and NR enzymatic activity in vivo in the cotton variety Fiber Max 989. The evaluated application rates were 0, 50, 100, and 150 kg/ha of N, using urea as a source (46% N) in a randomizedblock design with three replicates. At harvest, the maximum yield of seed cotton and the greatest accumulation of total foliar biomass through time was reached after applying 150 kg N/ha. The different N-application rates did not affect the components of cotton-fibre quality. The activity of endogenous NR was greater on plants where 150 kg N/ha were applied. The highest cotton yield and N contents were obtained on these plants. Therefore, the NR activity in vivo could be used as a bioindicator of the N nutritional level in cotton.     

Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment

The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.     

Germination of <i>Eryngium regnellii</i>: a major species for ecological restoration of plant-pollinator interactions in the Southern Pampas (Buenos Aires,Argentina)

Eryngium regnellii Malme belongs to the largest genera in the Apiaceae family, with 250 species worldwide and 65 represented in South America. It is a herbaceous species typical of hill plant communities, which, along with remnant grassland patches, are the most relevant natural habitats for the maintenance of diversity in the Southern Pampas. Eryngium regnellii is key to the maintenance of pollination mutualisms, being a generalist (displaying a diverse assemblage of pollinators) and ubiquitous species (present in all studied sierras). However, fragmentation of the Pampean landscape due to agricultural intensification has led to the loss of natural environments. Therefore, the reintroduction of E. regnellii in strategic places would facilitate the occurrence of wild pollinators, while favoring pollination services in the agroecosystem. The germination requirements of E. regnellii were studied because a better knowledge of the reproductive biology of this species would provide information relevant to its reproduction and reintroduction into degraded areas. Germination percentages and mean time to germination were evaluated, using one control and two pre-germination treatments: chemical scarification with sulfuric acid, and mechanical scarification with sand paper. Chemical scarified seeds did not germinate. Mechanically scarified and control seed groups showed no significant differences either in germination percentages (49% and 59% respectively) or in mean germination time (13 and 14 days, respectively). Results indicate that E. regnellii shows no physical dormancy, and does not require specific pre-germination treatments for germination under the studied laboratory conditions. The high germination capacity of E. regnellii, along with its ecological attributes, make it a potential species for restoring plant-pollinator interactions in the fragmented landscapes of the Southern Pampas.     

Relationship between DNA replicon size and SCE induction in BALB/c and BALB/Mo mouse lymphocytes

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions an increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.