Flow-cytometric detection of the RI alpha subunit of type I cAMP-dependent protein kinase in human cells

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.     

A new mathematical model to evaluate simazine removal in three different immobilized-biomass reactors

A new mathematical model based on the cinetical Langmuir equation is developed to interpret and predict the effectiveness of simazine (SZ) removal in immobilized-biomass reactor (IBR), to consider herbicide-support affinity (Cx), and herbicide-cell affinity (Cy). Three solid supports: sepiolite monolith, granular sepiolite, and alginate were used in pilot-scale reactors that were inoculated with Klebsiella planticola DSZ. The abiotic process was analysed by measuring the SZ sorption capacity of the reactor supports. Sepiolite monolith showed the maximum value for herbicide-support affinity (28.02+/-0.9%). The effectiveness of the biotic process was estimated considering the formation of biomass and SZ biodegradation. Granular sepiolite showed either higher affinity with SZ and viability rate (0.90) throughout the process, and SZ removal rate was 3.39+/-0.06 mg/h. The mathematical model presented in this paper provides useful insights into the interpretation of experimental data as well as prediction for the implementation of biological reactors.     

Biodegradation of oxadiazon by a soil isolated Pseudomonas fluorescens strain CG5: Implementation in an herbicide removal reactor and modelling

An oxadiazon-degrading bacterial, Pseudomonas strain CG5, was isolated from an agricultural contaminated soil. This strain CG5 was able to grow on 10mg of oxadiazon per l, yielding 5.18+/-0.2 mg of protein biomass mol(-1). GC-MS analyses of the metabolites from oxadiazon catabolism revealed its dehalogenation and degradation to form non-toxic end-products, cells were then immobilized by adsorption on a ceramic support to be used as biocatalysts in herbicide removal biofilm-reactor processes. Seventy-two per cent of the oxadiazon was removed, and the maximum specific substrate uptake rate was 10.63+/-0.5 microg h(-1) mg(-1) prot. A new mathematical model was developed to interpret and predict the behaviour of the bacteria and pollutants in a biofilm-reactor system, to consider biofilm structural and morphological properties.     

Therapeutic effects of phytosterols and phytostanols in cholesterolemia

Plant sterol and stanol esters are called "functional" compounds due to their hypocholesterolemic properties. The objective of this review is to update recent findings concerning the effect of phytosterols in the blood cholesterol, emphasizing the results from experimental and human studies. The hypocholesterolemic effect is observed with the intake of 2.5g/day of phytosterols or phytostanols. Daily intake, usually of stanols, for 4 weeks has shown to to be effective in lowering blood total- as well as LDL-cholesterol by about 10%. The mechanism of action in lowering blood cholesterol comes from their structural similarity to cholesterol, hence they act by competing with cholesterol at the luminal absorption site. The adverse effects of a high intake of phytosterols and phytostanols are the lower absorption of some liposoluble vitamins and antioxidants.     

N6-isopentenyladenosine affects cAMP-dependent microfilament organization in FRTL-5 thyroid cells

N6-Isopentenyladenosine (i6A), an adenosine and mevalonate derivative, inhibits, like adenosine, TSH-induced cAMP increase and its related events (I- uptake and DNA synthesis) in FRTL-5 cells. This inhibition is dose-dependent and is measurable at 10(-8) M. However, unlike adenosine, i6A prevents TSH-promoted microfilament disassembly. The effect of i6A on cytoskeletal structure is antagonized by pertussis toxin and could be assigned to its N6 substitution since it can be mimicked by other synthetic N6-adenosine derivatives. It is suggested that a step beyond cAMP is involved, since i6A prevents also microfilament disassembly induced by 8-bromo-cAMP. This is the first demonstration that an adenosine derivative, which is also an end-product of the isoprenoid pathway, affects cAMP-dependent microfilament organization.     

Impact of Magnetic Stimulation on Periodontal Ligament Stem Cells

The aim of this study was to evaluate the effect of a time-dependent magnetic field on the biological performance of periodontal ligament stem cells (PDLSCs). A Western blot analysis and Alamar Blue assay were performed to investigate the proliferative capacity of magnetically stimulated PDLSCs (PDLSCs MAG) through the study of the MAPK cascade (p-ERK1/2). The observation of ALP levels allowed the evaluation of the effect of the magnetic field on osteogenic differentiation. Metabolomics data, such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and ATP production provided an overview of the PDLSCs MAG metabolic state. Moreover, the mitochondrial state was investigated through confocal laser scanning microscopy. Results showed a good viability for PDLSCs MAG. Magnetic stimulation can activate the ERK phosphorylation more than the FGF factor alone by promoting a better cell proliferation. Osteogenic differentiation was more effectively induced by magnetic stimulation. The metabolic panel indicated significant changes in the mitochondrial cellular respiration of PDLSCs MAG. The results suggested that periodontal ligament stem cells (PDLSCs) can respond to biophysical stimuli such as a time-dependent magnetic field, which is able to induce changes in cell proliferation and differentiation. Moreover, the magnetic stimulation also produced an effect on the cell metabolic profile. Therefore, the current study demonstrated that a time-dependent magnetic stimulation may improve the regenerative properties of PDLSCs.     

Proton-conducting membranes from phosphotungstic acid-doped sulfonated polyimide for direct methanol fuel cell applications

A series of novel hybrid proton conducting membranes based on sulfonated naphthalimides and phosphotungstic acid (PTA) were prepared from N-Methyl Pyrrolidone (NMP) solutions. These hybrid organic-inorganic materials, composed of two proton-conducting components, have high ionic conductivities (9.3 × 10^?2 S cm^?1 at 60 °C, 15% PTA), and show good performance in H₂|O₂ polymer electrolyte membrane fuel cells (PEMFC), previously reported by us. Moreover, they have low methanol permeability compared to Nafion^®112. In this paper we describe, for the first time, the behaviour of these hybrid membranes as electrolyte in a direct methanol fuel cell (DMFC). The maximum power densities achieved with PTA doped sulfonated naphthalimide membrane, operating with oxygen and air, were 34.0 and 12.2 mW cm^?2, respectively; about the double and triple higher than those showed by the non-doped membrane at 60 °C.