Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)–atmospheric pressure chemical ionisation (APCI)–mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 μl of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17α-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC–APCI–MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25–1000 ng/ml (r > 0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.     

Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). β-amyloid (Aβ), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by Aβ is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that Aβ produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic Aβ1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric Aβ_1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized Aβ_1-42 compared to slices in a control experiment where no Aβ_1-42 exposure had occurred.Download video file.^{(55M, mp4)}     

Genetic predisposition and renal allograft failure: implication of non-HLA genetic variants

Renal allograft rejection or dysfunction often results in graft failure, and remains the major obstacle in the success of renal transplantation. Various immunological and nonimmunological factors are involved in allograft rejection. In addition to human leukocyte antigen loci, several genetically controlled molecules have been identified in recent years as playing important roles in the process of rejection. Genetic variants in genes encoding different T-helper (Th) type 1 and Th2 cytokines, chemokines and their receptors, growth factors, molecules of the renin-angiotensin system, enzymes of the homocysteine pathway, and proteins acting as substrates of immunosuppressive drugs impact on the success of engraftment and highlight the concept of genetic predisposition to allograft rejection. This review evaluates specific genetic variants and their functional roles in graft failure, with an emphasis on the latest methodologies available for genotyping, and appropriate strategies to enable them to become a tool of predictive and individualized medicine to ensure better transplant outcome.     

Correlation between EBV DNA and rearrangement and expression of Bcl-2 gene in aggressive non-Hodgkin's lymphoma

Previous in vitro studies have shown that bcl-2 expression can be induced by transfection of Epstein-Barr virus (EBV)-negative non-Hodgkin's lymphoma (NHL) cell lines with EBV. This induced expression of bcl-2 is important for the long survival of EBV-positive cells and might be a first step in tumorigenesis. The purpose of the present study was to investigate the possibility of similar correlation between bcl-2 expression and EBV infection in vivo in a cohort of patients with aggressive NHL, who were uniformly evaluated and treated with effective chemotherapy. The 42 patients included were 25-65 years old. None had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Fresh biopsied samples were obtained and stored frozen for analysis of bcl-2 gene rearrangement major break point and of EBV DNA by PCR. Bcl-2 protein expression was estimated by Western blot, and enzyme immunoassay. With a median follow-up of 30 months, overall survival (OS) and disease-free survival (DFS) were measured to determine the prognostic significance of these variables. Analyzable DNA was present in all samples, 24% demonstrating bcl-2 rearrangement and 33% showing EBV DNA. Patients with bcl-2 gene rearrangement tended to have shorter DFS, and OS than patients without translocation. Bcl-2 protein expression was not correlated to gene rearrangement and had no significant influence on survival. The presence of EBV DNA in NHL had no prognostic significance but was correlated to bcl-2 expression. EBV-positive tumors showed higher bcl-2 expression than EBV-negative tumors did. Our results suggest a role of EBV infection in inducing bcl-2 expression as a survival factor for EBV-positive cells.     

stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migrations of tracheal and mesodermal cells

Fibroblast growth factors (FGFs) bind to FGF receptors, transmembrane tyrosine kinases that activate mitogenic, motogenic, and differentiative responses in different tissues. While there has been substantial progress in elucidating the Ras-MAP kinase pathway that mediates the differentiative responses, the signal transduction pathways that lead to directed cell migrations are not well defined. Here we describe a Drosophila gene called stumps that is required for FGF-dependent migrations of tracheal and mesodermal cells. These migrations are controlled by different FGF ligands and receptors, and they occur by different cellular mechanisms: the tracheal migrations occur as part of an epithelium whereas the mesodermal migrations are fibroblast-like. In the stumps mutant, tracheal cells fail to move out from the epithelial sacs, and only rudimentary tracheal branches form. Mesodermal cells fail in their dorsal migrations after gastrulation. The stumps mutation does not block all FGF signaling effects in these tissues: both random cell migrations and Ras-MAP kinase-mediated induction of FGF-specific effector genes occurred upon ectopic expression of the ligand or upon expression of a constitutively activated Ras protein in the migrating cells. The results suggest that stumps function promotes FGF-directed cell migrations, either by potentiating the FGF signaling process or by coupling the signal to the cellular machinery required for directed cell movement.     

Role of nitric oxide in rotenone-induced nigro-striatal injury

Rotenone, a widely used pesticide, causes a syndrome in rats that mimics, both behaviorally and pathologically, the symptoms of Parkinson's disease. The present study evaluated the role of nitric oxide in rotenone-induced nigro-striatal injury. After administration of rotenone in rats for 40 days, there was a moderate but significant injury of the nigro-striatal pathway indicated by a 47% decrease in striatal dopamine levels and a 28% loss of substantia nigra tyrosine hydroxylase-immunopositive neurons. Furthermore, a significant (37%) increase in the number of cells positive for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the striatum was observed, accompanied by a 83% increase in nitric oxide synthase (NOS) activity and a significant increase in the production of 3-nitrotyrosine (3-NT). There was a significant increase (45%) in the optical density of NADPH-d staining and an increase (72%) in NOS activity in the substantia nigra. Moreover, administration of the neuronal NOS inhibitor 7-nitroindazole significantly attenuated the increased NOS activity and 3-NT production, and provided significant protection against rotenone-induced nigro-striatal injury. Our data suggest that chronic rotenone administration can lead to significant injury to the nigro-striatal system, mediated by increased generation of nitric oxide.     

Some toxicological characteristics of three venomous soft corals from the Red Sea

Three common Red Sea soft corals (Cnidaria: Anthozoa), Nephthea sp, Dendronephthya sp and Heteroxenia fuscescens sting humans. Nematocyst venoms of each animal are lethal to mice and hemolytic to human erythrocytes. However, these hemolysins are partially inhibited by known anti-hemolytic agents. Venoms and their gel chromatography-separated fractions have different dermonecrosis and vasopermeability potency in mouse skin. The venom of Heteroxenia fuscescens (Hf) was more lethal (LD50: 0.7 mg/kg), with one prominent 97-kDa protein fraction (LD50: 0.55 mg/kg). Hf venom was more hemolytic, more dermonecrotic, and had more vasopermeable factors than that of the two other species. SDS polyacrylamide gel electrophoresis of soft coral whole venoms and fractions showed different protein molecular masses ranging from 200 to less than 6 kDa. High IgG titers were assayed from venom-sensitized mice blood sera. Enzyme-linked immunosorbent assays (ELISA) marked significant immunological cross-reaction between the studied soft coral venoms and their bioactive fractions.     

Peroxynitrite and the regulation of Na(+),K(+)-ATPase activity by angiotensin II in the rat proximal tubule.

NO reacts spontaneously with superoxide to produce the potent oxidant peroxynitrite. Studies were designed to examine the role of NO-derived oxidants and peroxynitrite on the regulation of Na(+),K(+)-ATPase activity by angiotensin II (ANG II) freshly isolated rat proximal tubules. At picomolar concentrations ANG II stimulates Na(+),K(+)-ATPase activity, but at nanomolar concentrations stimulation is lost. Superoxide dismutase (SOD) was used to examine the role of superoxide and deferoxamine (DFO) and uric acid (UA) were used to examine the role of peroxynitrite. SOD (200 U/mL, 5-min preincubation) restored the stimulatory effect of ANG II (1.31 +/- 0.08-fold; n = 4; P < 0.05 compared to 10(-7) M alone), suggesting a role for superoxide. DFO (100 microm, 5-min preincubation) also restored the stimulatory effect of ANG II (1.40 +/- 0.08-fold; n = 4; P < 0.05, compared to 10(-7) M alone), as did UA (1.22 +/- 0.07-fold; n = 5; P < 0.05, compared to 10(-7) M alone). The NO synthesis inhibitor, N-monomethyl-L-arginine (L-NMMA, 2 mM; 5-min preincubation), also unmasked a stimulatory effect of ANG II at 10(-7) M (1.4 +/- 0.1-fold; n = 7; P < 0.05, compared to 10(-7) M alone). The generation of peroxynitrite was further evidenced by the formation of 3-nitrotyrosine (3-NT). 3-NT increased 3.5-fold in tubules exposed to ANG II (10(-7) M) (0.0054 +/- 0.0019 3-NT/100 tyrosines for control and 0.019 +/- 0.0058 3-NT/100 tyrosines for ANG II, P < .05; n = 4) and L-NMMA prevented the increase. These data suggest that peroxynitrite signaling participates in the regulation of renal of Na(+),K(+)-ATPase activity.     

A pseudocystic lipoma in the European eel (Anguilla anguilla)

This is the first report of lipoma in the European eel Anguilla anguilla. In a single eel that was obtained from a polyculture fish farm in northern Egypt, a yellowish swelling (10 mm in diameter) was observed near the tip of the lower lip. The tumor was composed of mature lipocytes that occasionally fused into an unlined cystic space.     

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens– a valuable medicinal plant

A rapid and efficient protocol for the large‐scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15‐day‐old aseptic seedlings is described. Of the three different cytokinins, 6‐benzyladenine (BA), kinetin (Kin) and 2‐isopentenyl adenine (2‐iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half‐strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α‐naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole‐3‐butyric acid (IBA). The in vitro‐raised plantlets with well‐developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.