Combination of optison with ultrasound and electroporation increases albumin and thrompoietin transgene expression whilst elongation factor promoter prolongs its duration

Hypoalbuminaemia and thrombocytopaenia are two clinical problems frequently encountered in patients with chronic liver failure or cancer following treatment with chemotherapy. The current study was designed to assess the magnitude and duration of thrombopoietin and albumin transgene expression hoping to increase the production of albumin and platelets. Immunocompetent and immunocompromised (nude) mice were injected intramuscularly with plasmids expressing either human serum albumin or human thrombopoietin. The therapeutic expression cassette of the plasmids was driven by either CMV or elongation factor 1- promoters respectively. In order to achieve muscle specific expression both gene constructs included the myosin light chain enhancer. The experiment was conducted in a group of mice which were injected with the transgene plasmid either in normal saline or plasmid followed by electroporation, ultrasound, optison and a combination of all three to increase transgene expression. The result showed that plasmids with the CMV promoter induced the highest transgenic expression lasting for one week whilst plasmids with the elongation factor 1-alpha promoter produced a weaker expression lasting for a longer and more stable duration of expression up to 3 months in both immunocompetent and nude mice. The combination of electroporation and ultrasound with Optison TM provided the highest transgene expression. We concluded that it would be possible to increase albumin and platelets production by an intramuscular injection of plasmids expressing human albumin and thromopoietin. A combination of electroporation and ultrasound with Optison TM can increase their expression

Nifedipin ublažava djelovanje kokaina na enzimsku aktivnost u mozgu i jetri te smanjuje njegovo izlučivanje putem mokraće

The aim of this study was to see how nifedipine counters the effects of cocaine on hepatic and brain enzymatic activity in rats and whether it affects urinary excretion of cocaine. Male Wistar rats were divided in four groups of six: control, nifedipine group (5 mg kg-1 i.p. a day for five days); cocaine group (15 mg kg-1 i.p. a day for five days), and the nifedipine+cocaine group. Twenty-four hours after the last administration, we measured neuronal nitric oxide synthase (nNOS) activity in the brain and cytochrome P450 quantity, ethylmorphine-N-demethylase, and anilinehydroxylase activity in the liver. Urine samples were collected 24 h after the last cocaine and cocaine+nifedipine administration. Urinary cocaine concentration was determined using the GC/MS method. Cocaine administration increased brain nNOS activity by 55 % (p<0.05) in respect to control, which indicates the development of tolerance and dependence. In the combination group, nifedipine decreased the nNOS activity in respect to the cocaine-only group. In the liver, cocaine significantly decreased and nifedipine significantly increased cytochrome P450, ethylmorphine-N-demethylase, and anilinehydroxylase in respect to control. In combination, nifedipine successfully countered cocaine effects on these enzymes. Urine cocaine excretion in the cocaine+nifedipine group significantly dropped (by 35 %) compared to the cocaine-only group. Our results have confirmed the effects of nifedipine against cocaine tolerance and development of dependence, most likely due to metabolic interactions between them.Cilj je ovoga istraživanja bio utvrditi kako nifedipin ublažava djelovanje kokaina na enzimsku aktivnost u mozgu i jetri Wistar štakora te utječe li na njegovo izlučivanje putem mokraće. Mužjaci su podijeljeni u četiri skupine po šest jedinki: kontrolnu skupinu, nifedipinsku skupinu koja je pet dana intraperitonealno primala nifedipin u dozi od 5 mg kg-1; skupinu koja je pet dana primala kokain u dozi od 15 mg kg-1 na dan te skupinu koja je zajedno primala nifedipin i kokain u odgovarajućim dozama. Dvadeset i četiri sata nakon posljednje doze izmjerena je enzimska aktivnost sintaze dušičnoga oksida (nNOS) u mozgu, razina citokroma P450 te aktivnosti enzima etilmorfi n-N-demetilaze i anilinhidroksilaze u jetri štakora. Uzorci mokraće prikupljeni su 24 sata nakon posljednje doze kokaina odnosno kombinacije nifedipina i kokaina. Koncentracija kokaina u mokraći izmjerena je s pomoću vezanog sustava plinske kromatografi je i spektrometrije masa. Kokain je povećao aktivnost nNOS-a u mozgu za 55 % (p<0,05) u odnosu na kontrolnu skupinu, što upućuje na stvaranje tolerancije i ovisnosti. U kombiniranoj skupini nifedipin je značajno smanjio aktivnost nNOS-a u odnosu na skupinu koja je primila samo kokain. Kokain je značajno snizio, a nifedipin značajno povisio razinu citokroma P450 u jetri te aktivnost etilmorfi n-N-demetilaze i anilinhidroksilaze u odnosu na kontrolnu skupinu. U kombiniranoj skupini nifedipin je uspješno ublažio djelovanje kokaina na aktivnost spomenutih enzima. Izlučivanje kokaina putem mokraće u kombiniranoj skupini bilo je značajno manje (35 %) nego u skupini koja je primala samo kokain. Ovi rezultati potvrđuju da nifedipin štiti od djelovanja kokaina i stvaranja ovisnosti, najvjerojatnije zbog interakcija u metabolizmu dvaju spojeva

Extensive retreat of Greenland tidewater glaciers 2000-2010

Overall mass loss from the Greenland ice sheet nearly doubled during the early 2000s resulting in an increased contribution to sea-level rise, with this step-change being mainly attributed to the widespread frontal retreat and accompanying dynamic thinning of tidewater glaciers. Changes in glacier calving-front positions are easily derived from remotely sensed imagery and provide a record of dynamic change. However, ice-sheet-wide studies of calving fronts have been either spatially or temporally limited. In this study multiple calving-front positions were derived for 199 Greenland marine-terminating outlet glaciers with width greater than 1 km using Landsat imagery for the 11-year period 2000–2010 in order to identify regional seasonal and inter-annual variations. During this period, outlet glaciers were characterized by sustained and substantial retreat summing to more than 267 km, with only 11 glaciers showing overall advance. In general, the pattern of mass loss detected by GRACE (Gravity Recovery and Climate Experiment) and other measurements is reflected in the calving record of Greenland glaciers. Our results suggest several regions in the south and east of the ice sheet likely share controls on their dynamic changes, but no simple single control is apparent

Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii

Activation of the steroid and xenobiotic receptor, SXR, induces apoptosis in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>The steroid and xenobiotic receptor, SXR, is an orphan nuclear receptor that regulates metabolism of diverse dietary, endobiotic, and xenobiotic compounds. SXR is expressed at high levels in the liver and intestine, and at lower levels in breast and other tissues where its function was unknown. Since many breast cancer preventive and therapeutic compounds are SXR activators, we hypothesized that some beneficial effects of these compounds are mediated through SXR.</p> <p>Methods</p> <p>To test this hypothesis, we measured proliferation of breast cancer cells in response to SXR activators and evaluated consequent changes in the expression of genes critical for proliferation and cell-cycle control using quantitative RT-PCR and western blotting. Results were confirmed using siRNA-mediated gene knockdown. Statistical analysis was by t-test or ANOVA and a P value ≤ 0.05 was considered to be significant.</p> <p>Results</p> <p>Many structurally and functionally distinct SXR activators inhibited the proliferation of MCF-7 and ZR-75-1 breast cancer cells by inducing cell cycle arrest at the G1/S phase followed by apoptosis. Decreased growth in response to SXR activation was associated with stabilization of p53 and up-regulation of cell cycle regulatory and pro-apoptotic genes such as p21, PUMA and BAX. These gene expression changes were preceded by an increase in inducible nitric oxide synthase and nitric oxide in these cells. Inhibition of iNOS blocked the induction of p53. p53 knockdown inhibited up-regulation of p21 and BAX. We infer that NO is required for p53 induction and that p53 is required for up-regulation of cell cycle regulatory and apoptotic genes in this system. SXR activator-induced increases in iNOS levels were inhibited by siRNA-mediated knockdown of SXR, indicating that SXR activation is necessary for subsequent regulation of iNOS expression.</p> <p>Conclusion</p> <p>We conclude that activation of SXR is anti-proliferative in p53 wild type breast cancer cells and that this effect is mechanistically dependent upon the local production of NO and NO-dependent up-regulation of p53. These findings reveal a novel biological function for SXR and suggest that a subset of SXR activators may function as effective therapeutic and chemo-preventative agents for certain types of breast cancers.</p

Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry.

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed

Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product.

Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5- (and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 x 10(-8) m s-1. Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40 degrees C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s-1 was calculated. Hydrolysis of cFDA (at 40 degrees C) in cell extracts was described by Michaelis-Menten kinetics with an apparent Vmax and Km of 12.3 nmol.min-1.mg of protein-1 and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed