Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome–human serum albumin association

Fluorescence correlation spectroscopy was used to measure the diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label for liposomes and HSA as well. For decomposing the fluorescence intensity autocorrelation function (ACF) into components corresponding to a liposome population, HSA and MP, we used a maximum entropy procedure that computes a distribution of diffusion times consistent with the ACF data. We found that a simple parametric non-linear fit with a discrete set of decay components did not converge to a stable parameter set. The distribution calculated with the maximum entropy method was stable and the average size of the particles calculated from the effective diffusion time was in good agreement with the data determined using the discrete-component fit.     

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [³H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([³H]Met-enkephalin-Arg⁶-Phe⁷), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K _D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K _D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n _H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [³H]Met-enkephalin-Arg⁶-Phe⁷ binding reflects a κ₂- and/or δ-subtype specificity of the heptapeptide. Binding to a κ₁ and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg⁶-Phe⁷ in frogs cannot be ruled out. The [³H]Met-enkephalin-Arg⁶-Phe⁷ binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.     

The newly synthesized plant growth regulator <Emphasis Type="Italic">S</Emphasis>-methylmethionine salicylate may provide protection against high salinity in wheat

High salinity is one of the major environmental factors limiting the productivity of crop species worldwide. Improving the stress tolerance of cultivated plants and thus increasing crop yields in an environmentally friendly way is a crucial task in agriculture. In the present work the ability of a new derivative, S-methylmethionine-salicylate (MMS), to improve the salt tolerance of wheat plants was tested parallel with its related compounds salicylic acid and S-methylmethionine. The results show that while these compounds are harmful at relatively high concentration (0.5 mM), they may provide protection against high salinity at lower (0.1 mM) concentration. This was confirmed by gas exchange, chlorophyll content and chlorophyll-a fluorescence induction measurements. While osmotic adjustment probably plays a critical role in the improved salt tolerance, neither Na or K transport from the roots to the shoots nor proline synthesis are the main factors in the tolerance induced by the compounds tested. MMS, S-methylmethionine and Na-salicylate had different effects on flavonol biosynthesis. It was also shown that salt treatment had a substantial influence on the SA metabolism in wheat roots and leaves. Present results suggest that the investigated compounds can be used to improve salt tolerance in plants.     

TPPP/p25 Promotes Tubulin Acetylation by Inhibiting Histone Deacetylase 6

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of α-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of α-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 μm concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

SELDI-TOF MS detection of urinary hepcidin

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.     

Evaluation of Drug Release From Coated Pellets Based on Isomalt,Sugar, and Microcrystalline Cellulose Inert Cores

The objective of the present study was to investigate the effect of the pellet core materials isomalt, sugar, and microcrystalline cellulose on the in vitro drug release kinetics of coated sustained-release pellets as well as to evaluate the influence of different ratios of polymethacrylate copolymers exhibiting different permeability characteristics on the drug release rate. For characterization of the drug release process of pellets, the effect of osmolality was studied using glucose as an osmotically active agent in the dissolution medium. The pellet cores were layered with diclofenac sodium as model drug and coated with different ratios of Eudragit® RS30D and Eudragit® RL30D (ERS and ERL; 0:1 and 0.5:0.5 and 1:0 ratio) in a fluid bed apparatus. Physical characteristics such as mechanical strength, shape, and size proved that the inert cores were adequate for further processing. The in vitro dissolution tests were performed using a USP Apparatus I (basket method). The results demonstrated that, besides the ratio of the coating polymers (ERS/ERL), the release mechanism was also influenced by the type of starter core used. Sugar- and isomalt-type pellet cores demonstrated similar drug release profiles.     

Long‐term survival and diversification of an endemic Melitaea species in mountains of Iran and adjacent areas

Disjunct distribution patterns regularly resulted in the separation of different genetic lineages in glacial refugia. Recent patterns of survival and expansion have been often revealed by climatic niche modelling. We used the combination of genetic markers, geometric morphometry and climatic niche modelling to clear up the taxonomy and reconstruct the potential range of an endemic Iranian, taxonomically disputed Melitaea species in climatically different epochs. Our results show that this species (Melitaea abbas Gross and Ebert 1975, comb. n. = M. zagrosi Tóth and Varga syn. nova) is clearly separated from all taxa of the Melitaea phoebe species group and only occurs in Iran and Azerbaijan but were also predicted for some adjacent regions. Molecular markers and distribution modelling show consistently that this species should have had a long‐term survival in this area, and its range could have been slightly larger during the last glacial maximum than currently. Based on the studied molecular markers, three main groups in M. abbas can be recognized: those of steppic area of Azerbaijan, western Iran and northeastern Iran. Each group is characterized by own mitochondrial haplotypes, but also a high level of genetic diversity appears in the central part of the distribution area (Zagros Mts.).