Secreting oviduct epithelial cells of Coturnix coturnix japonica (QOEC) and changes to their proteome after nonviral transfection

The quail oviduct (Coturnix c. japonica) is a natural candidate avian bioreactor, while the secretive quail oviduct epithelial cells (QOECs) are potential in vitro producers of recombinant proteins and vaccines. In view of the need for highly performing and transformable cell lines, QOEC may potentially act as an alternative bioreactor platform to the existing ones, for example, to the Chinese hamster ovary. The aim of this work was to characterize QOECs and their response to nucleofection with a nonviral plasmid DNA carrying the human interferon-α 2a gene (hIFNλ2a), in vitro. Primary QOEC cultures from laying quails (10-15 weeks old) were characterized by their proliferation rate, doubling time, and multilineage differentiation. Electroporation to cell nuclei (nucleofection) was used to deliver nonviral plasmid DNA containing a reporter GFP and hIFN under the ovalbumin promoter. The posttransfection analysis included polymerase chain reaction, Western blot analysis, and liquid chromatography coupled to tandem mass spectrometry. QOEC showed a typical epithelial characteristic in a primary 2D monolayer culture system and retained secretive potential up to the first passage. QOEC showed differentiation into osteoblastic lineage after stimulation. The nucleofection mean efficiency was low (2.3%). Differences of up to 10% in the proteomic profiles between nontransfected and transfected QOEC were found, the most important of these were related to the absence of keratins and cell-adhesion proteins in the transfected QOEC. Concluding, with the practical information provided here, QOEC have the potential to serve as an avian secreting cellular platform. QOEC may be further transformed to cell lineage to meet the requirement for a stable, electrocompetent, and transfectable model. The first proteomic comparison of QOEC delivered in this study showed, in the majority, a stable proteome of the nontransfected vs transfected QOEC.     

A calcium-binding protein with similarity to serum albumin localized to the ER-Golgi network and cell walls of spinach (Spinacia oleracea)

Using polyclonal antibodies raised against human serum albumin (HSA), a 70-kDa microsomal protein with an isoelectric point of approximately 6.5 was detected in spinach ( Spinacia oleracea L.). The protein was purified by selective ammonium sulfate precipitation and anion exchange HPLC. The protein shared 100% identity with the first 15 amino acids at the NH₂ terminus of HSA, including the X-X-H amino acid region, which was identified in HSA as being responsible for binding of copper, zinc, indole derivatives and calcium. Blue staining of the protein with the cationic carbocyanine dye 'Stains-all' and ⁴⁵Ca overlay following SDS-PAGE also suggest that the 70-kDa plant protein binds calcium. The protein reacted positively with carbohydrate specific thymol stain, and the carbohydrates associated with the protein were identified by gas chromatography-mass spectrometry (GC-MS) as galactose and galacturonic acid. The 70-kDa plant protein was present in the detergent-poor phase following Triton X-114 extraction of the microsomal proteins. Cell fractionation using continuous sucrose gradients showed that the protein is present in membrane fractions with high activity of endoplasmic reticulum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probed with anti-HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted to the apoplast of cortex cells in plants. Localization and binding properties suggest that the plant protein identified in the present study may participate in secretion processes, possibly involved with the transport of precursors required for cell-wall synthesis.     

The Influence of Spike Rate and Stimulus Duration on Noradrenergic Neurons

We model spiking neurons in locus coeruleus (LC), a brain nucleus involved in modulating cognitive performance, and compare with recent experimental data. Extracellular recordings from LC of monkeys performing target detection and selective attention tasks show varying responses dependent on stimuli and performance accuracy. From membrane voltage and ion channel equations, we derive a phase oscillator model for LC neurons. Average spiking probabilities of a pool of cells over many trials are then computed via a probability density formulation. These show that: (1) Post-stimulus response is elevated in populations with lower spike rates; (2) Responses decay exponentially due to noise and variable pre-stimulus spike rates; and (3) Shorter stimuli preferentially cause depressed post-activation spiking. These results allow us to propose mechanisms for the different LC responses observed across behavioral and task conditions, and to make explicit the role of baseline firing rates and the duration of task-related inputs in determining LC response.     

Binary mixtures of (N-phosphonomethyl)-glycine with new aminophosphonates

The potential biological activity of binary mixtures of some new organophosphorous compounds, aminoalkane- and aminofluorenephosphonates, with (N-phosphonomethyl)-glycine (glyphosate, PMG) was studied. The inhibition of growth of wheat (Triticum aestivum) induced by individual compounds and their equimolar mixtures with PMG was a measure of that activity. The experiments were expected to show if the new compounds exhibited good biological activity to be used for agrochemical applications and if this activity can be improved when they are used in mixtures with glyphosate which is the active component of the well-known herbicide Roundup. The results obtained show that aminofluorenephosphonates inhibited wheat growth when used in micromolar concentrations. Thus, their efficiency can be compared to that of PMG. The efficiency of aminoalkanephosphonates was one order of magnitude weaker. The measure of the efficiency was the effective concentration inhibiting wheat growth by 50% (EC50). The most demanded interaction, i.e., a synergistic was observed for only one of binary mixtures of the compounds studied with PMG. Mostly they showed antagonistic or strong antagonistic interactions. Some of them were of the additive type. Such results exclude the possibility of potential use of all the compounds studied in binary mixtures with phosphonomethylglycine, especially as the mentioned synergistic interaction found was rather weak. The influence of structural features of anminophosphonates on the results obtained is discussed.     

Impact of 1,5-disubstituted tetrazole ring on chelating ability of delta-selective opioid peptide

Complex formation between Cu(II) and three tetrazole analogues of opioid peptide-deltorphin I has been investigated. In potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been established the thermodynamic stability, speciation and structure of Cu(II) complexes with Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (L1), Tyr-Psi(CN4)-Gly-Phe-Asp-Val-Val-Gly-NH2 (L2), Tyr-Gly-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L3) and Tyr-D-Ala-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L4). The site of the insertion of tetrazole moiety Psi(CN4) into the peptide sequences has critical impact on their co-ordination ability. Comparison of the binding ability of the tetrazole analogues reveals that around physiological pH region the L3 and L4 are more effective ligands for copper(II) than L(1) and L(2). The peptide conformation changes achieved by Cu(II) co-ordination may be essential for binding of the tetrazole deltorphins at the opiate receptors.     

The development of new bicyclic pyrazole-based cytokine synthesis inhibitors

4-Aryl-5-pyrimidyl-based cytokine synthesis inhibitors of TNF-alpha production, which contain a novel bicyclic pyrazole heterocyclic core, are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell-based assay and against human p38 alpha MAP kinase in an isolated enzyme assay. The X-ray crystal structure of a bicyclic pyrazole inhibitor co-crystallized with mutated p38 (mp38) is presented.     

Role of epigenetic DNA alterations in the pathogenesis of systemic lupus erythematosus

Epigenetic alternations in genomic DNA encompass cytosine methylation in cytosine and guanine (CpG) dinucleotide islands, which are usually extended in the promoter and first exon of genes. The DNA methylation is carried out by DNA methyltransferases (DNMT) and it serves as an epigenetic method of gene expression modulation. The epigenetic alternations in genomic DNA have been implicated in the development of malignant and autoimmune diseases. The epigenetic aberration in regulatory DNA sequences may also be responsible for the emergence of changes in the immune system in patients with systemic lupus erythematosus (SLE). The agents 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) belong to inhibitors of methyltransferase. These compounds affect the methylation level of promoter sequences and cause phenotypic changes in peripheral blood mononuclear cells (PBMC), which are similar to those observed in PBMC of SLE patients. The lack of methylcytosine in CpG dinucleotides may be responsible for the antigenic properties of microbial DNA. The presence of low-apoptotic methylated DNA fragments has been identified in plasma of SLE patients. These DNA fragments exhibit antigenic properties and may elicit the humoral response responsible for the flare of SLE. The low methylation of CpG residues in the regulatory sequences may also contribute to the elevated expression of human endogenous retroviruses (HERVs) in PBMC of SLE patients. The HERV components exhibit a profound similarity with nuclear antigens and may be responsible for the enhancement of the production of anti-antinuclear antibodies (ANA). Recent advances in the investigation of epigenetic DNA changes have formed the basis of improved understanding of etiopathogenesis of SLE, which may thereby facilitate improvement in therapeutic principles of this disease.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

Direct separation, identification and quantification of epimers 22R and 22S of budesonide by capillary gas chromatography on a short analytical column with Rtx-5 stationary phase

The conditions for separation, identification and quantitative determination of epimers 22R and 22S of budesonide by capillary gas chromatography (GC) with FID detection and two various sample injection methods, namely split-splitless and cool on-column, were established. In analysis helium as carrier gas and Rtx-5 capillary column of 7 m in length along with stationary phase Crossbond 5% diphenyl-95% dimethyl polysiloxane were used. The individual epimers were identified under specified conditions by using standard samples of different declared concentration of each epimer under investigation: (1) 51.2% of epimer 22R and 47.3% of epimer 22S, and (2) 95.1% of 22R and 4.4% of 22S, as well as Pulmicort, a preparation containing micronized budesonide as an active substance. It seems that good parameters of preliminary validation achieved by the proposed methods can confirm its suitability for quantitative analysis purpose. The retention times obtained for epimers 22R and 22S, depending on injection technique are about 7.7 and. 8.3 min for split and, approx. 10.3 and 10.9 min for cool on-column. The limits of detection and quantitation are 5.7 and 6.2 ng, for 22R respectively, and 4.3 and 4.8 ng for 22S. The linearity is maintained for concentrations ranging from 0.01 to 0.20 mg/ml. The quantitative analysis features of repeatability, high precision and accuracy confirmed by the obtained results and its statistical evaluation.     

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.