Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF

The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 microL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 microL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010-1.0 pmol/microL; lopinavir and ritonavir, 0.0025-0.50 pmol/microL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/microL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025-0.010 pmol/microL).     

Morphine modulation of thrombospondin levels in astrocytes and its implications for neurite outgrowth and synapse formation

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple μ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by μ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFβ1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of μ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.     

The subcellular distribution of calnexin is mediated by PACS-2

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.     

Combined and selective miR-21 silencing and doxorubicin delivery in cancer cells using tailored DNA nanostructures

MicroRNAs play an important role in tumorigenesis and, among them, miR-21 is found to be aberrantly up-regulated in various tumors. The tumor-associated antigen, folate receptor alpha is a GPI-membrane protein overexpressed in many malignant tumors of epithelial origin, including ovarian and cervical cancers. Covalently bound octahedral DNA nanocages were functionalized with folate molecules and utilized as scaffolds to engineer four sequestering units with a miR-21 complementary sequence for obtaining biocompatible Fol-miR21-NC non-toxic nanostructures, to be able to selectively recognize folate receptor alpha-overexpressing cancer cells and sequester the oncogenic miR-21. qPCR assays showed that Fol-miR21-NCs reduce the miR-21 expression up to 80% in cancer cells in the first 2 days of treatment. Functional assays demonstrated that miR-21 sequestering leads to up-regulation of miR-21 tumor suppressor targets (i.e., PTEN and Pdcd4), reduction in cancer cell migration, reduction in proliferation, and increase in cell death. Fol-miR21-NCs can be efficiently loaded with the chemotherapeutic agent doxorubicin. Co-delivery of anti-miR-21 and doxorubicin showed additive cytotoxic effects on tumor cells, paving the way for their use as selective nucleic acid drugs.Subject terms: Nucleic-acid therapeutics, Cancer therapy     

Paracrine role of Sertoli cells

Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.     

β-Endorphin Is a Potent Inhibitor of Thymidine Incorporation into DNA via μ and κ-Opioid Receptors in Fetal Rat Brain Cell Aggregates in Culture

Abstract: Thymidine incorporation into DNA was inhibited dose-dependently by β-endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by μ (cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide) or k (norbinaltorphimine) antagonists. Complete blockade of the β-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie–Hofstee analysis revealed that β-endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, β-endorphin decreased thymidine incorporation with an IC₅₀ of 0.7 n M . Truncated and N -acetylated β-endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that β-endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.     

Methodology for the implementation of monitoring plans with different spatial and temporal scales of plant protection products residues in water bodies based on site-specific environmental pressures assessments

Abstract

The awareness of plant protection products residues causing problems in water bodies is increasing more and more. A lot of effort is being made by countries in investigating the situation of diffuse pesticide pollution. This article illustrates a new methodology developed for the implementation of monitoring plans for plant protection products residues in rivers, lakes and groundwater, at river basin scale, based on an operational workflow which, by integrating different databases, let to evaluate site-specific environmental pressures affecting the definition of the related monitoring networks. It follows that sampling and analytical activities, carried out in the monitoring network nodes, not only are functional to water bodies ecological and chemical quality status assessment but are able to highlight possible compromises of environmental balance in agro-ecosystems, deriving from plant protection products use, through the application of environmental modeling able to bring out evolutive trends. This information would allow the Administrators to take increasingly effective initiatives both in the field of controls and authorizations for particular substances, in the light of the negative effects shown, and in the field of spatial planning, being able to dispose of the necessary knowledge in order to take safeguard measures before a certain evolutionary process of degradation becomes irreversible.     

Association between Low‐Molecular Weight Apolipoprotein(a) Isoforms and Obesity in Italian Women

Objective: Low‐molecular weight (MW) apolipoprotein(a) [apo(a)] isoforms are closely associated with an increased incidence of atherothrombotic disease, prevalence of which is higher in obese individuals, particularly in women. The hypothesis of this study was to assess whether there are differences in the distribution of apo(a) phenotypes between obese patients and healthy controls. Research Methods and Procedures: One hundred three obese Italian women (BMI ≥ 30.0 kg/m²) were enrolled in the study, and apo(a) phenotyping was performed in all subjects. The prevalence of low‐MW apo(a) isoforms, detected in plasma samples of our obese women, was compared with that found in a control group of 84 normal‐weight, never‐obese (BMI < 25.0 kg/m²), age‐matched women. Results: The distribution of apo(a) isoforms in the population of obese women was significantly different from that found in normal‐weight female subjects. In particular, the percentage of subjects in the obese group with at least one apo(a) isoform of low MW was significantly higher than that in the control group (51.4% vs. 32.1%, p = 0.0079). Discussion: Our results seem to suggest the possibility that small‐sized apo(a) isoforms may be used together with other traditional risk factors to better assess the overall predisposition to atherothrombotic disease in obese women.