Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

Nitroheterocyclic compounds (NC) were candidate drugs proposed for Chagas disease chemotherapy. In this study, we investigated the complexation of hydroxymethylnitrofurazone (NFOH), a potential antichagasic compound, with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), Hydroxypropyl-beta-cyclodextrin (HP-beta-CD), Dimethyl-beta-cyclodextrin (DM-beta-CD) and gamma-cyclodextrin (gamma-CD) by fluorescence spectroscopy and molecular modeling studies. Hildebrand-Benesi equation was used to calculate the formation constants of NFOH with cyclodextrins based on the fluorescence differences in the CDs solution. The complexing capacity of NFOH with different CDs was compared through the results of association constant according to the following order: DM-beta-CD > beta-CD > alpha-CD > HP-beta-CD > gamma-CD. Molecular modeling studies give support for the experimental assignments, in favor of the formation of an inclusion complex between cyclodextrins with NFOH. This is an important study to investigate the effects of different kinds of cyclodextrins on the inclusion complex formation with NFOH and to better characterize a potential formulations to be used as therapeutic options for the oral treatment of Chagas disease.

Coupling Between GABA(A)-R Ligand-Binding Activity and Membrane Organization in beta-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with beta-cyclodextrin (beta-CD). The mere pre-incubation (PI) at 37A degrees C accompanying the beta-CD treatment was an underlying source of perturbations increasing [H-3]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4A degrees C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the beta-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not beta-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with beta-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, beta-CD is not completely eliminated from the system through centrifugation washings. It was concluded that beta-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual beta-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

Production of cyclodextrins by CGTase from Bacillus clausii using different starches as substrates

Cyclodextrins ( CDs) are cyclic oligasaccharides composed by D- glucose monomers joined by alpha- 1,4-D glicosidic linkages. The main types of CDs are alpha-,beta-and gamma-CDs consisting of cycles of six, seven, and eight glucose monomers, respectively. Their ability to form inclusion complexes is the most important characteristic, allowing their wide industrial application. The physical property of the CD-complexed compound can be altered to improve stability, volatility, solubility, or bio-availability. The cyclomaltodextrin glucanotransferase ( CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into CD molecules. In this work, the CGTase produced by Bacillus clausii strain E16 was used to produce CD from maltodextrin and different starches ( commercial soluble starch, corn, cassava, sweet potato, and waxy corn starches) as substrates. It was observed that the substrate sources influence the kind of CD obtained and that this CGTase displays a beta- CGTase action, presenting a better conversion of soluble starch at 1.0%, of which 80% was converted in CDs. The ratio of total CD produced was 0: 0.89: 0.11 for alpha/beta/gamma. It was also observed that root and tuber starches were more accessible to CGTase action than seed starch under the studied conditions.

Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

Photophysical properties of a photocytotoxic fluorinated chlorin conjugated to four beta-cyclodextrins

A meso-tetrakis(pentafluorophenyl)-chlorin with the reduced pyrrole ring linked to an isoxazolidine ring (FC) has been conjugated to four beta-cyclodextrins (CDFC). The CDFC exhibits excellent water solubility and is a potent photosensitizer towards proliferating NCTC 2544 human keratinocytes. The study by conventional steady state absorption and fluorescence spectroscopies and by time-resolved femto- and nanosecond laser flash spectroscopies suggests that in ethanol and pH 7 buffer the beta-cyclodextrins embed the highly hydrophobic tetrakis(pentafluorophenyl)-chlorin macrocycle and strongly interact with the chlorin rings in the singlet and triplet manifolds. In these solvents, femtosecond spectroscopy suggests that the conjugate undergoes a rapid relaxation in the upper excited singlet states induced by photochemical and/or conformation change(s) at a rate of about 5 ps(-1) to fluorescent states whose lifetime is similar to 8 ns. This interaction is destroyed upon addition of Triton X100 to buffer. Both FC and CDFC strongly fluoresce (Phi(F) similar to 0.5) in micelles. Similar behavior is observed at the triplet level. In ethanol and water, the initial transient triplet state absorbance decays within 1-3 mu s yielding a longer lived triplet with spectral properties indistinguishable from that of original difference absorbance spectra. The determination of the molar absorbance in the 440-460 nm region (similar to 35 000 M(-1) cm(-1)) leads to an estimate of similar to 0.2 for the triplet formation quantum yield of FC in toluene and of FC and CDFC in Triton X100 micelles. Quenching of the CDFC triplets by dioxygen in buffer produces (1)O(2) in a good yield consistent with the effective photocytotoxicity of the chlorin-cyclodextrins conjugate towards cultured NCTC 2544 human keratinocytes. By contrast, FC which aggregates in buffer produces little if any (1)O(2).

High-Density Lipoprotein Inhibits the Uptake of Modified Low-Density Lipoprotein and the Expression of CD36 and Fc gamma RI

Aim: Modified low-density lipoprotein (mLDL), mainly upon oxidative and enzymatic modification, is the major atherogenic lipoprotein. Conversely, high-density lipoprotein (HDL) is considered anti-atherogenic because of its ability to remove cholesterol. The aim of this work was to analyze both the influence of HDL on the uptake of mLDL and the expression of CD36 and Fc gamma I receptors on monocytic cell lines during cell differentiation. Methods: Uptake of fluorescein isothiocyanate (FITC)-conjugated LDL and FITC-conjugated mLDL, i.e., copper-oxidized LDL (oxLDL) or trypsin enzyme modified LDL (enzLDL), was analyzed, as well as the expression of CD36 and Fc gamma RI in THP-1 and U937 cells, using flow cytometry. Results: HDL inhibited the uptake of mLDL, which varied in degree depending on the cell line or type of mLDL. Further, HDL rapidly decreased CD36 and Fc gamma RI involved in the uptake of mLDL. Conclusions: We demonstrate that modified LDL promotes specific LDL receptor-independent uptake by monocytic cell lines, and that the uptake of LDL and enzLDL is less than that of oxLDL. In this process, HDL diminishes the uptake of LDL or mLDL, which may involve the down-regulation of receptors (CD36 and Fc gamma I). This regulatory process represents another way by which HDL can be anti-atherogenic and it depends on the type of modification of LDL and the stage of differentiation of monocytes to macrophages.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12-secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Predictive values of aspartate aminotransferase and gamma-glutamyl transferase for the hepatic accumulation of copper in cattle and buffalo

Ten cattle and 10 buffalo were divided into 2 groups (control [n = 8] and experimental [n = 12]) that received daily administration of copper. Three hepatic biopsies and blood samples were performed on days 0, 45, and 105. The concentration of hepatic copper was determined by spectrophotometric atomic absorption, and the activities of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) were analyzed. Regression analyses were done to verify the possible existing relationship between enzymatic activity and concentration of hepatic copper. Sensitivity, specificity, accuracy, and positive and negative predictive values were determined. The serum activities of AST and GGT had coefficients of determination that were excellent predictive indicators of hepatic copper accumulation in cattle, while only GGT serum activity was predictive of hepatic copper accumulation in buffalo. Elevated serum GGT activity may be indicative of increased concentrations of hepatic copper even in cattle and buffalo that appear to be clinically healthy. Thus, prophylactic measures can be implemented to prevent the onset of a hemolytic crisis that is characteristic of copper intoxication.

Populations III.1 and III.2 gamma-ray bursts: constraints on the event rate for future radio and X-ray surveys

Aims. We calculate the theoretical event rate of gamma-ray bursts (GRBs) from the collapse of massive first-generation (Population III; Pop III) stars. The Pop III GRBs could be super-energetic with the isotropic energy up to E(iso) greater than or similar to 10(55-57) erg, providing a unique probe of the high-redshift Universe. Methods. We consider both the so-called Pop III.1 stars (primordial) and Pop III.2 stars (primordial but affected by radiation from other stars). We employ a semi-analytical approach that considers inhomogeneous hydrogen reionization and chemical evolution of the intergalactic medium. Results. We show that Pop III.2 GRBs occur more than 100 times more frequently than Pop III.1 GRBs, and thus should be suitable targets for future GRB missions. Interestingly, our optimistic model predicts an event rate that is already constrained by the current radio transient searches. We expect similar to 10-10(4) radio afterglows above similar to 0.3 mJy on the sky with similar to 1 year variability and mostly without GRBs (orphans), which are detectable by ALMA, EVLA, LOFAR, and SKA, while we expect to observe maximum of N < 20 GRBs per year integrated over at z > 6 for Pop III.2 and N < 0.08 per year integrated over at z > 10 for Pop III.1 with EXIST, and N < 0.2 for Pop III.2 GRBs per year integrated over at z > 6 with Swift.

Comparative Analysis of Activation Phenotype, Proliferation, and IFN-gamma Production by Spleen NK1.1(+) and NK1.1(-) T Cells During Plasmodium chabaudi AS Malaria

The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-gamma production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1(+) and NK1.1(-) T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4(+)NK1.1(+)TCR-alpha beta(+), CD8(+)NK1.1(+)TCR-alpha beta(+), and CD4(+)NK1.1(-)TCR-alpha beta(+) cell numbers per spleen, where a higher increment was observed for NK1.1(+) T cells compared to NK1.1(-) T cells. According to the ability to recognize the CD1d-alpha-GalCer tetramer, CD4(+)NK1.1(+) cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1(+) T cells and around 30% of NK1.1(-) T cells showed an experienced/activated (CD44(HI)CD69(HI)CD122(HI)) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-gamma production by CD4(+)NK1.1(+) cells peaked at day 4 p.i., the IFN-gamma response of CD4(+)NK1.1(-) cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin(+) cells in CD8(+)NK1.1(+) cells compared to CD8(+)NK1.1(-) cells. These results indicate that spleen NK1.1(+) and NK1.1(-) T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-gamma production.

Observation of double electron-positron pair production by gamma rays reexamined

An experiment was conducted to observe triple- and quadruple-escape peaks, at a photon energy equal to 6.128 MeV, in the spectra recorded with a high-purity Ge detector working in coincidence with six bismuth germanate detectors. The peak intensities may be explained having recourse to only the bremsstrahlung cascade process of consecutive electron-positron pair creation; i.e., the contribution of simultaneous double pair formation (and other cascade effects) is much smaller. The experimental peak areas are in reasonably good agreement with those predicted by Monte Carlo simulations done with the general-purpose radiation-tran sport code PENELOPE.

Search for the rare decay K(L)->pi(0)pi(0)gamma

The KTeV E799 experiment has conducted a search for the rare decay K(L)->pi(0)pi(0)gamma via the topology K(L)->pi(0)pi(0)(D)gamma (where pi(0)(D)->gamma e(+)e(-)). Because of Bose statistics of the pi(0) pair and the real nature of the photon, the K(L)->pi(0)pi(0)gamma decay is restricted to proceed at lowest order by the CP conserving direct emission (DE) of an E2 electric quadrupole photon. The rate of this decay is interesting theoretically since chiral perturbation theory predicts that this process vanishes at level O(p(4)). Therefore, this mode probes chiral perturbation theory at O(p(6)). In this paper we report a determination of an upper limit of 2.43x10(-7) (90% CL) for K(L)->pi(0)pi(0)gamma. This is approximately a factor of 20 lower than previous results.

eta ->gamma gamma decay width via the Primakoff cross section

Incoherent eta photoproduction in nuclei is evaluated at forward angles within 4 to 9 GeV using a multiple scattering Monte Carlo cascade calculation with full eta-nucleus final-state interactions. The Primakoff, nuclear coherent and nuclear incoherent components of the cross sections fit remarkably well previous measurements for Be and Cu from Cornell, suggesting a destructive interference between the Coulomb and nuclear coherent amplitudes for Cu. The inelastic background of the data is consistently attributed to the nuclear incoherent part, which is clearly not isotropic as previously considered in Cornell's analysis. The respective Primakoff cross sections from Be and Cu give Gamma(eta ->gamma gamma)=0.476(62) keV, where the quoted error is only statistical. This result is consistent with the Particle Data Group average of 0.510(26) keV and in sharp contrast (similar to 50%) with the value of 0.324(46) keV obtained at Cornell.