Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Background

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Methods

Targeting elements (biotin‐labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19?), Granta 519 (CD3?/ CD19+), and J.RT3‐T3.5 (CD3?/CD19?)] using the EGFP gene as a reporter gene and anti‐CD3 and anti‐CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry.

Results

A significant selectivity of gene delivery was observed, since the anti‐CD3 immunopolyplex worked only in Jurkat cells while the anti‐CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3? cell mixture with anti‐CD3 immunopolyplexes showed up to 16‐fold more transfection in CD3+ than in CD3? cells. Several non‐specific transfection reagents showed poor or no transfection activity.

Conclusion

It is concluded that immunopolyplex is a good non‐viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) – the streptavidin–polyplex backbone remaining intact – thereby conferring high versatility. Copyright © 2002 John Wiley & Sons, Ltd.

     

Soil-vegetation relationships in cerrado (Brazilian savanna) and semideciduous forest, Southeastern Brazil

Several studies pointed out soil properties as the prime determinant ofcerrado (the Brazilian savanna) physiognomies, and a gradient from campocerrado (a shrub savanna) to cerradão (a tallwoodland) has been correlated with a soil fertility gradient. Based on thishypothesis, we investigated soil-vegetation relationships in thePé-de-Gigante Reserve (São Paulo State,SoutheasternBrazil). We randomly distributed 10 quadrats (10 × 10 m) oneach ofthe following physiognomies: campo cerrado, cerradosensu stricto, cerradão, andseasonal semideciduous forest, previously defined by the analysis of satelliteimages (LANDSAT-5). We sampled the woody individuals with stem diameter> 3 cm at soil level, identifying their species. In each quadrat, wecollectedsoil samples at the depths of 0–5, 5–25, 40–60, and80–100 cm, and determined pH, K, Ca, Mg, P, Al, H + Al, basesaturation, aluminium saturation, cation exchange capacity, and percentage ofsand, clay and loam. Obtained data were submitted to a canonical correspondenceanalysis (CCA) and to a detrended correspondence analysis (DCA). Our resultsshowed a clear distinction between semideciduous forest and the cerradophysiognomies, based in soil parameters. The former was related to higherconcentrations of cations and clay in the soil, while the latter was related tohigher concentrations of exchangeable aluminium in the soil surface. The threecerrado physiognomies – campo cerrado, cerradosensu stricto, and cerradão– could not be distinguished considering plant density and the analysedsoil features.     

Gap-phase regeneration in a semideciduous mesophytic forest, south–eastern Brazil

The study was carried out in ten natural canopy gaps in the SantaGenebra County Reserve (22°4945S, 47°0633W)in the county of Campinas, São Paulo State, Brazil. The size and canopyopenness of the gaps were studied using hemispherical photographs. Thevegetation survey included all shrubs and trees with height 0.50m in the gaps interiors and all the individuals with PBH(perimeterat breast height) 15 cm in a 3 m surroundingborder of the gaps. The similarity among the gaps and among their surroundingareas was assessed by the Jaccard similarity index and by cluster analyzes. Thegap size varied from 20.09 to 468 m², with apredominance of small gaps. The families with the greatest species richness inthe gaps were Rutaceae, Rubiaceae and Euphorbiaceae. The species with thegreatest number of individuals in the gaps were Coffeaarabica L., Hybanthus atropurpureus (St. Hil.)Taub. and Actinostemon klotschii (Muell. Arg.) Pax, allwidely distributed in the understorey. Shade-tolerant species (late secondaryspecies) predominated in function of the predominance of small gaps. The highnumber of species found in the gaps reflects the importance of thesedisturbances in the maintenance of species diversity in the studied forest.     

The effect of high dose digoxin on cytokines in healthy dogs

BACKGROUND: Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are pro-inflammatory cytokines, causing myocardial dysfunction and a negative inotropic effect. The drugs used to treat heart failure affect the production of cytokines. Digoxin, on which this study was focused, is one of the drugs for the treatment of heart failure. AIM: The present study was designed to examine the early effects of high doses of digoxin on the production of cytokines in healthy dogs. METHODS: Digoxin was given parenterally to dogs at 0.15 mg/kg. IL-1beta and TNF-alpha production and levels of digoxin in the serum were measured 0, 12, 24, 48, and 72 h following administration of digoxin. RESULTS: As the levels of serum digoxin taken at 12, 24, 48, and 72 h of administration were considered significantly high compared with preceding values (p < 0.001), no notable change in serum IL-1beta and TNF-alpha levels was observed. CONCLUSIONS: These results suggest that high doses of digoxin do not cause a significant cytokine production in heart muscle in the early phase.     

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.     

Guanidinium and aminoimidazolinium derivatives of N-(4-piperidyl)propanamides as potential ligands for mu opioid and I2-imidazoline receptors: synthesis and pharmacological screening

Derivatives of N-(1-phenethyl-4-piperidyl)propanamides incorporating guanidinium and 2-aminoimidazolinium groups have been prepared by a synthetic approach involving first introduction of a spacer between the piperidine and the functional group by reductive amination of piperidinone. The formation of each of these functional groups was carried out using N-N'-di(tert-butoxycarbonyl)thiourea and 2-methylthioimidazolinium iodide, respectively. These structures have been designed to incorporate two pharmacologic goals into one entity. Radioligand binding assays have been used to study their affinity for opioid (mu, delta and kappa) and I2-imidazoline receptors. Two of them, 10 and 16, showed high affinity for mu opioid receptors and functionally they had moderate analgesic properties in the hot plate and writhing tests. The in vitro studies on guinea pig ileum (GPI) indicated that both compounds are mu opioid agonists. In what concerns I2-imidazoline receptor activity, these derivatives showed low affinity around 6 to 7 times less than idazoxan.     

Xanthones as inhibitors of growth of human cancer cell lines and their effects on the proliferation of human lymphocytes in vitro

Twenty-seven oxygenated xanthones have been assessed for their capacity to inhibit in vitro the growth of three human cancer cell lines, MCF-7 (breast cancer), TK-10 (renal cancer) and UACC-62 (melanoma). The effect of these xanthones on the proliferation of human T-lymphocytes was also evaluated. Differences on their potency towards the effect on the growth of the human cancer cell lines as well as on the proliferation of human T-lymphocytes can be ascribed to the nature and positions of the substituents on the xanthonic nucleus.     

Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.