The role of miRNA-221 and miRNA-126 in patients with benign metastasizing leiomyoma of the lung: an overview with new interesting scenarios

Molecular Biology Reports - Benign metastasizing leiomyoma (BML) is a rare disease characterized by extrauterine benign leiomyomatosis in patients with a previous or concomitant history of uterine...     

Enhanced cytokine secretion from primary macrophages due to Dectin-1 mediated uptake of CpG DNA/β-1,3-glucan complex

Unmethylated CpG sequences (CpG DNA) can induce Th1 response and thus become a potential immunotherapeutic agent. A key step in the treatment is to transport CpG DNA to its receptor TLR9 located in the endocytosis pathway of target immune cells. For the effective transport, we prepared a novel complex from a β-1,3-glucan schizophyllan (SPG) and CpG DNA, and administered the complex to murine peritoneal macrophages that had been previously activated by thioglycollate and expressed a major β-1,3-glucan receptor Dectin-1 on the cellular surface. Flow cytometric analysis and microscopic observation showed that the complex was taken up by the macrophage through Dectin-1 mediated pathway. Indeed, ELISA demonstrated that IL-12 production was increased sigmoidally with increasing SPG/CpG DNA ratio in the complexation, and reached the maximum at the SPG-rich composition. In the present work, we describe a new approach to deliver CpG DNA to immune cells by use of a β-1,3-glucan/DNA complex.     

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.     

Exercise preconditioning modulates genotoxicity induced by doxorubicin in multiple organs of rats

The aim of this study was to investigate the effects of exercise in multiple organs of rats treated with doxorubicin. Male adult Wistar rats were distributed into the following groups: sedentary + NaCl; exercise + NaCl; sedentary + doxorubicin; and exercise + doxorubicin. Animals were sacrificed 2 days following injections. Central fragments from heart, liver, and kidney were collected and minced in 0.9% NaCl being cellular suspensions used for the single-cell gel (comet) assay. The results showed that exercise was able to prevent genotoxicity induced by doxorubicin in heart cells. By contrast, exercise was not able to prevent genotoxicity induced by doxorubicin in liver cells. The same occurred to kidney cells, i.e. no statistically significant differences (p > 0.05) were found when compared with groups not exposed to doxorubicin. Taken together, our results support the idea that exercise could contribute to the protective effect against genotoxicity induced by doxorubicin in heart cells.     

Enhanced cytokine secretion owing to multiple CpG side chains of DNA duplex

Unmethylated CpG sequences (CpG ODN) stimulate Toll-like receptor 9 (TLR9) to activate innate immunity. We made DNA duplexes from poly(dT)320 and CpG ODN with (dA)40 attached at the 3' end. Circular dichroism and gel electrophoresis indicated that the CpG parts turned outward from the duplex. When we changed the CpG ODN/poly(dT) molar ratio, the amount of IL-12 secreted from J774A.1 cells (murine macrophage-like) reached the maximum at the compositions with two to four CpG portions in one duplex, while the maximum loading was eight CpG ODNs per one poly(dT)320. When the residual free dT parts were hybridized with its control GpC ODN with (dA)40 tail or just (dA)40, the maximum disappeared and the secretion increased with increasing the CpG molar ratio. These results indicated that there is a particular DNA higher-order structure to activate TLR9 more efficiently than single CpG ODN.     

A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.     

Lysine residues located on the surface of human plasma high-density lipoprotein particles

Normal human plasma high-density lipoprotein (HDL) was reacted with trinitrobenzenesulfonate (TNBS). The time course of the reaction of intact HDL in phosphate buffer (pH 8.6) at 20 degrees C was biphasic, having a transition plateau. Both the first phase and the second phase reactions followed pseudo-first-order kinetics, and their rate constants at 20 degrees C were 4.6 X 10(-2) and 4.9 X 10(-3)/min, respectively. The activation energies of these reactions estimated from Arrhenius plots were 22.9 kcal/mol for the first phase and 20.9 kcal/mol for the second phase. The frequency factors, however, were markedly different from each other. On the other hand, the biphasic kinetic curves could not be observed when HDL was reacted with TNBS after delipidation, heat denaturation or Triton X-100 treatment. The rate constants for TNBS reactions of apolipoprotein HDL and heat-denatured HDL were essentially the same, 1.0 X 10(-2)/min, and those for HDL and apolipoprotein HDL in Triton X-100 were also the same, 3.57 X 10(-3)/min at 20 degrees C. Furthermore, fluorodinitrobenzene (FDNB), a typical penetrating chemical probe, reacted with intact HDL, indicating a nonbiphasic nature. These observations suggest that two reacting classes of lysine residues in intact HDL particles can be distinguished by the biphasic TNBS reaction, and that at the first phase of the reaction TNBS modifies selectively the accessible lysine residues exposed to surrounding aqueous environments. On this basis, intact HDL3 was reacted with TNBS at 20 degrees C for 80 min and it could be considered that at least 10 of 22 primary amino groups of apolipoprotein A-I and 10 of 18 lysine residues of apolipoprotein A-II were exposed on the surface of HDL3 particles.     

Characterization of the lateral interaction between human erythrocyte spectrin subunits.

A technique in which the subunits of human erythrocyte spectrin were immobilized on a nitrocellulose membrane was developed to study which domains of the subunit are able to bind to the counterpart subunit. The limited tryptic digestion of the isolated alpha and beta subunits of human erythrocyte spectrin produced eight fragments in the alpha subunits and nine fragments in the beta subunit. Four fragments of the beta (80, 60, 44, and 18 kDa) and two of the alpha (82 and 33 kDa) bound to alpha and beta subunits which were immobilized on nitrocellulose membrane strips, respectively. The binding affinities of all the fragments to the subunits, however, were remarkably lower than that of the mother proteins. The titration of fluorescence anisotropy of N-(1-anilinonaphthyl-4)maleimide which was covalently attached to the subunit by the trypsin-digested fraction of the counterpart subunit also indicate weak binding of the fragments even in solution. These findings suggest that the high-affinity binding of the alpha subunit to the beta subunit to form spectrin alpha beta dimer occurs only when the binding domains are arrayed along the polypeptide chains at the appropriate positions on the subunits.