Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PMNs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P. brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H(2)O(2), the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PMNs killing activity and H(2)O(2) release were inhibited. Together, our results show the importance of PMNs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.     

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AimsCardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na⁺/K⁺-ATPase.Main methodsInhibition of Na⁺/K⁺-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released.Key findingsAnalyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na⁺/K⁺-ATPase protomer.SignificanceConsidering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.     

Maternal-Fetal Distribution of Calcium,Iron, Copper,and Zinc in Pregnant Teenagers and Adults

Adolescence is marked by intensive growth and development. When pregnancy occurs during this period of the mother’s growth, there is an increase in her nutritional needs. The aim of this study was to determine the levels of calcium, iron, copper, and zinc in maternal plasma, the placenta, and in the cord plasma of pregnant teenagers and adults. A total of 80 sets of maternal plasma, placentas, and cord plasma (40 from teenagers and 40 from adults) were analyzed using synchrotron radiation total reflection X-ray fluorescence. The levels of calcium, copper, and zinc in the maternal and cord plasma from teenagers were not significantly different than those from adults. Iron levels in the teenagers maternal and cord plasma were higher than in the adults. All of the minerals analyzed were present at higher levels in the placentas from adults than those from teenagers. However, the low quantities of placental calcium, iron, copper, and zinc in the teenagers do not compromise the levels of these minerals in the cord plasma. Future research regarding the placental transport of these minerals is recommended to investigate the efficiency of mechanisms of transfer of these minerals in pregnant teenagers.     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

Prebiotic processes in planetary atmospheres

Within 40 years of experimental studies in prebiotic chemistry, most of the building blocks of the living systems have been synthesized in plausible conditions of the primitive Earth. The starting ingredients correspond to two complementary classes: volatile organics, and their non volatile oligomers. They may have been formed in the atmosphere on the primitive Earth and/or imported by extra-terrestrial sources. Organic chemistry is involved in meteorites, comets, in the giant planets and several of their satellites. Again this chemistry presents the two complementary aspects. In particular, with a dense reduced atmosphere rich in organic compounds in gas and aerosol phases, Titan appears as a natural laboratory for studying prebiotic chemistry at a planetary scale.     

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.     

X-ray diffraction characterization of the dense phases formed by nucleosome core particles

Multiple dense phases of nucleosome core particles (NCPs) were formed in controlled ionic conditions (15-160 mM monovalent salt, no divalent ions), under osmotic pressures ranging from 4.7 x 10(5) to 2.35 x 10(6) Pa. We present here the x-ray diffraction analysis of these phases. In the lamello-columnar phase obtained at low salt concentration (<25 mM), NCPs stack into columns that align to form bilayers, kept separated from one another by a layer of solvent. NCPs form a monoclinic lattice in the plane of the bilayer. For high salt concentration (>50 mM), NCPs order into either a two-dimensional columnar hexagonal phase or into three-dimensional orthorhombic (quasi-hexagonal) crystals. The lamellar and hexagonal (or quasi-hexagonal) organizations coexist in the intermediate salt range; their demixing requires a long time. For an applied pressure P = 4.7 10(5) Pa, the calculated NCPs concentration ranges from approximately 280 to 320 mg/ml in the lamello-columnar phase to 495 to 585 mg/ml in the three-dimensional orthorhombic phase. These concentrations cover the concentration of the living cell.