GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

GABAB receptors are expressed in human aortic smooth muscle cells and regulate the intracellular Ca2+ concentration

The aim of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter gamma-aminobutyric acid (GABAB), in human aortic smooth muscle cells (HASMCs), and to explore if altering receptor activation modified intracellular Ca(2+) concentration ([Ca(2+)]i) of HASMCs. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABABR1 and GABABR2 in cultured HASMCs. Immunohistochemistry was used to localize the two subunits in human left anterior descending artery (LAD). The effects of the GABAB receptor agonist baclofen on [Ca(2+)]i in cultured HASMCs were demonstrated using fluo-3. Both GABABR1 and GABABR2 mRNA and protein were identified in cultured HASMCs and antibody staining was also localized to smooth muscle cells of human LAD. 100 μM baclofen caused a transient increase of [Ca(2+)]i in cultured HASMCs regardless of whether Ca(2+) was added to the medium, and the effects were inhibited by pre-treatment with CGP46381 (selective GABAB receptor antagonist), pertussis toxin (a Gi/o protein inhibitor), and U73122 (a phospholipase C blocker). GABAB receptors are expressed in HASMCs and regulate the [Ca(2+)]i via a Gi/o-coupled receptor pathway and a phospholipase C activation pathway

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells

Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)–mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine “threshold” exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTORSer2448 (~1.4-fold; P < .04), 4E-BP1 Thr37/46 (~1.9-fold; P < .001), and rpS6Ser235/6 (~2.3-fold; P < .001). However, only p70S6kThr389 exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6kThr389 follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.

Development and application of Palygorskite porous ceramsite in a biological aerated filter (BAF)

Novel filter Palygorskite porous ceramsite (PC) was prepared using Palygorskite clay, poreforming material sawdust, and sodium silicate with a mass ratio of 10:2:1 after sintering at 700°C for 180 min. PC was characterized with X-ray diffraction, X-ray fluorescence, scanning electron microscopy, elemental, and porosimetry. PC had a total porosity of 67% and specific surface area of 61 m2/g. In order to assess the usefulness of PC as a medium for biological aerated filters (BAF), PC and (commercially available ceramsite) CAC were used to treat wastewater city in two laboratory-scale upflow BAFs. The results showed that the reactor containing PC was more efficient than the reactor containing CAC in terms of total organic carbon (TOC), ammonia nitrogen (NH3-N), and the removal of total nitrogen (TN) and phosphorus (P). This system was found to be more efficient at water temperatures ranging from 20 to 26°C, an air–water (A/W) ratio of 3:1, dissolved oxygen concentration >4.00 mg/L, and hydraulic retention time (HRT) ranging from 0.5 to 7 h. The interconnected porous structure produced for PC was suitable for microbial growth, and primarily protozoan and metazoan organisms were found in the biofilm. Microorganism growth also showed that, under the same submerged culture conditions, the biological mass in PC was significantly higher than in CAC (34.1 and 2.2 mg TN/g, respectively). In this way, PC media can be considered suitable for the use as a medium in novel biological aerated filters for the simultaneous removal of nitrogen and phosphorus.

Unmodified silver nanoparticles for rapid analysis of the organophosphorus pesticide, dipterex, often found in different waters

A novel, uncomplicated and rapid method of analysis for organophosphorus (OP) pesticides was researched and developed using the important, common OP, dipterex, as a typical example. The basis of the method involved the citrate-capped silver nanoparticles (citrate-capped AgNPs) and Acetylthiocholine (ATCh). The latter compound can be catalyzed by Acetylcholinesterase (AChE) to form thiocholine (TCh), which induces the aggregation of AgNPs. Correspondingly, the color of AgNPs in solution changes from bright yellow to pink, and the UV–vis characteristic absorption peak of AgNPs at about 400 nm decreases; simultaneously, a new absorption band appears at about 520 nm. Irreversible inhibition of AChE activity caused by dipterex, prevents the aggregation of AgNPs. Thus, a UV–vis spectrophotometric method was developed for the analysis of dipterex. The absorbance ratio A396 nm/A520 nm was found to be linearly related to the concentration of dipterex in the range of 0.25–37.5 ng mL−1 with a detection limit of 0.18 ng mL−1. This method was used successfully to analyse dipterex in spiked, different water samples.

Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 μg in 2002 to 500-5000 μg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 μg/ml and 6.3 μg/mg dry cell mass respectively) and the lowest was for LB (2.8 μg/ml and 3.3 μg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 μg/ml and 11.2 μg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

Creation of protein loaded biodegradable microparticles via ultrasonic atomization suitable for nasal delivery

Improved biopharmaceutical delivery may be achieved via the use of biodegradable microspheres as delivery vehicles. Biodegradable microspheres offer the advantages of maintaining sustained protein release over time whilst simultaneously protecting the biopharmaceutical from degradation. Particle samples produced by ultrasonic atomization were studied in order to determine a feed stock capable of producing protein loaded poly-ε-caprolactone (PCL) particles suitable for nasal delivery (i.e., less than 20 μm). A 40 kHz atomization system was used with a 6 mm full wave atomization probe. The effect of solids percent, feed flow rate, volumetric ratio of the polymer stock to the protein stock, and protein concentration in the protein stock on particle size characteristics were determined. It was shown that feed stocks containing 100 parts of 0.5 or 1.0% w/v PCL in acetone with one part 100 mg ml -1 BSA and 15 mg ml -1 PVA produced particles with a mass moment diameter (D[4,3]) of 13.17 μm and 9.10 μm, respectively in addition to displaying high protein encapsulation efficiencies of 93 and 95%, respectively. The biodegradable PCL particles were shown to be able to deliver encapsulated protein in vitro under physiological conditions.

Exploration of the fundamental equilibrium behaviour of calcium exchange with weak acid cation resins

This study evaluated the complexity of calcium ion exchange with sodium exchanged weak acid cation resin (DOW MAC-3). Exchange equilibria recorded for a range of different solution normalities revealed profiles which were represented by conventional “L” or “H” type isotherms at low values of equilibrium concentration (Ce) of calcium ions, plus a superimposed region of increasing calcium uptake was observed at high Ce values. The loading of calcium ions was determined to be ca. 53.5 to 58.7 g/kg of resin when modelling only the sorption curve created at low Ce values,which exhibited a well-defined plateau. The calculated calcium ion loading capacity for DOWMAC-3 resin appeared to correlate with the manufacturer's recommendation. The phenomenon of super equivalent ion exchange (SEIX) was observed when the “driving force” for the exchange process was increased in excess of 2.25 mmol calcium ions per gram of resin in the starting solution. This latter event was explained in terms of displacement of sodium ions from sodium hydroxide solution which remained in the resin bead following the initial conversion of the as supplied “H+” exchanged resin sites to the “Na+” version required for softening studies. Evidence for hydrolysis of a small fraction of the sites on the sodium exchanged resin surface was noted. The importance of carefully choosing experimental parameters was discussed especially in relation to application of the Langmuir–Vageler expression. This latter model which compared the ratio of the initial calcium ion concentration in solution to resin mass, versus final equilibrium loading of the calcium ions on the resin; was discovered to be an excellent means of identifying the progress of the calcium–sodium ion exchange process. Moreover, the Langmuir–Vageler model facilitated standardization of various calcium–sodium ion exchange experiments which allowed systematic experimental design.

Suppression of cluster ions during rapidly increasing particle number concentration events in the environment

We show that the cluster ion concentration (CIC) in the atmosphere is significantly suppressed during events that involve rapid increases in particle number concentration (PNC). Using a neutral cluster and air ion spectrometer, we investigated changes in CIC during three types of particle enhancement processes – new particle formation, a bushfire episode and an intense pyrotechnic display. In all three cases, the total CIC decreased with increasing PNC, with the rate of decrease being greater for negative CIC than positive. We attribute this to the greater mobility, and hence the higher attachment coefficient, of negative ions over positive ions in the air. During the pyrotechnic display, the rapid increase in PNC was sufficient to reduce the CIC of both polarities to zero. At the height of the display, the negative CIC stayed at zero for a full 10 min. Although the PNCs were not significantly different, the CIC during new particle formation did not decrease as much as during the bushfire episode and the pyrotechnic display. We suggest that the rate of increase of PNC, together with particle size, also play important roles in suppressing CIC in the atmosphere.