Enhancement of murine erythropoiesis in vitro by a porcine kidney extract.

We have examined the effect of porcine kidney extract (PKE) on the growth of erythroid progenitor-derived colonies in a methylcellulose culture. The addition of PKE resulted in an enhancement of burst-forming-unit-erythroid (BFU-E)-derived colonies, and the enhancement of the colony was also observed in a low concentration of erythropoietin (Epo) and fetal calf serum (FCS). The activity of PKE on BFU-E was compared with the erythroid growth factors, such as recombinant murine interleukin-3 (IL-3), recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) and recombinant human granulocyte colony-stimulating factor (G-CSF) which are all well known to stimulate BFU-E growth. IL-3 showed a potent burst-promoting activity (BPA), but GM-CSF and G-CSF did not enhance BFU-E growth in our culture conditions. In the cultures supplemented with Epo, the rapid loss of BFU-E was prevented with IL-3; however, PKE alone did not prevent the disappearance of BFU-E. These results suggest that PKE possesses a BPA-like activity which is considered an enhancement of BFU-E.     

Comparative studies of the colony-promoting activity of porcine kidney extract with several interleukins and colony-stimulating factors.

Porcine kidney extracts (PKE) possess colony-promoting activity (CPA) which stimulates primitive hematopoietic cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), but PKE itself does not stimulate colony formation on murine bone marrow cells. We have compared the CPA of PKE with that of recombinant cytokines or CSFs such as interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), GM-CSF and macrophage colony-stimulating factor (CSF-1). All of these factors were less potent than PKE. Furthermore, the combinations of IL-1 alpha or PKE with G-CSF, GM-CSF, IL-3 or IL-6 were examined in the presence of one of these factors such as CSF. It is found that PKE acts synergistically with G-CSF, GM-CSF, IL-3 and IL-6, showing enhancement ratios of 10, 2.5, 4.2 and 30, respectively. The combination of IL-1 alpha resulted in poor colony formation in contrast with those of PKE, except for CSF-1. These results suggest that the CPA of the factor(s) in PKE differ from the cytokines and CSFs tested in this study, and is significantly affected by various types of CSF.     

Two polyene amides produced by genetically modified Streptomyces diastaticus var. 108

Streptomyces diastaticus var. 108, a newly isolated strain, was recently characterized as a producer of two polyene macrolide antibiotics (rimocidin and CE-108), and the biosynthetic gene cluster was partially characterized. When the producer strain was genetically modified by transformation with some engineered SCP2*-derived vectors carrying the ermE gene, two previously uncharacterized macrolides were detected in the fermentation broth of the recombinant strain and chemically characterized as the amides of the parental polyene carboxylic acids. The biological activity and some in vitro toxicity assays showed that this chemical modification resulted in pharmaceuticals with improved biological properties compared with the parental products.     

Phage-display selection of a human single-chain fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G(M3)-carbohydrate antigen

Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.     

Synthesis and Biological Evaluation of Several Dephosphonated Analogues of CMP‐Neu5Ac as Inhibitors of GM3‐Synthase

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP‐Neu5Ac congeners and their anti ‐ GM3‐synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3‐synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.     

CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells

A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.     

Effect of Fish Bone Bioactive Peptides on Oxidative,Inflammatory and Pigmentation Processes Triggered by UVB Irradiation in Skin Cells

In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-α), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.     

Induction of differentiation of human leukemia cells by various combinations of cytokines and low-molecular-weight inducers

To explore agents for differentiation therapy of leukemias, various combinations of cytokines and low-molecular-weight inducers were examined for differentiation-inducing activity toward three kinds of human leukemia-derived cell lines. The strongest differentiation inducing activity on promyelocytic HL60 cells and histiocytic U937 cells was obtained by combining recombinant tumor necrosis factor (rTNF), interferon-gamma (IFN-gamma), retinoic acid (RA), and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). For myeloblastic ML1 cells, the combination of rTNF, IFN-gamma, and RA had the strongest differentiation-inducing activity.     

Iminosugar C-Glycosides Work as Pharmacological Chaperones of NAGLU,a Glycosidase Involved in MPS IIIB Rare Disease**

Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C-glycosides mimicking N-acetyl-D-glucosamine and bearing various pseudo-anomeric substituents of both α- and β-configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non-functionalized and wrongly configured β-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration.     

油菜花粉十二肽和类似物的合成及性质的研究

用固相法合成了首次从油菜花粉中提取的十二肽，为进一步研究其结构与活性的关系，设计并合成了它的７个肽片段和５个类似物，并通过ＦＴＩＲ和ＣＤ谱的测定与分析，初步讨论了它们在固体状态和不同溶剂中的结构问题，同进，用肿瘤坏死因子ＴＮＦ实验，Ｔ－淋巴细胞转化实验和Ｅ－玫瑰花结实验测定了它们的生理活性，结果提示该十二肽和它的某些片段具有增强ＴＮＦ抗肿瘤细胞和在体外能促进猪胸腺细胞成熟的作用。     

Interaction of fibrinogen with nanosilica

Interaction of human plasma fibrinogen (HPF) with fumed nanosilica A-300 in a phosphate buffer solution (PBS) was studied using ¹H NMR spectroscopy with layer-by-layer freezing-out of bulk and interfacial water in the temperature range of 210–273 K, TSDC (90 < T < 265 K), adsorption, FTIR, and UV spectroscopy methods. An increase in concentration of HPF in the PBS leads to a decrease in amounts of structured water (frozen at T < 273 K) because of coagulation of HPF molecules. Addition of nanosilica to the HPF solution strongly reduces the amounts of structured water because of adsorption interaction of HPF molecules with silica nanoparticles, self-association of HPF molecules, formation of denser packed hybrid agglomerates with HPF and silica, and lastly, because of conformational changes of HPF. A monolayer adsorption capacity of A-300 corresponds to 156 mg of HPF per gram of silica. The FTIR and UV spectra show that the HPF adsorption on silica leads to structural changes of the protein molecules. These changes and formation of hybrid HPF/A-300 aggregates can increase the rate of clotting that is of importance on nanosilica application as a component of tourniquet preparations.      

Liposomalization of hydroxyphenyl fluorescein as a reagent for detecting highly reactive oxygen species

It has been shown that lifestyle-related diseases and aging are related to reactive oxygen species (ROS), and many studies have reported on the direct detection of ROS. The topical fluorescence reagent 2,7-dichlorodihydrofluorescein (DCDHF) is used to measure oxidation. However, there are problems regarding its stability, and its similar sensitivity to ROS cannot be easily distinguished. In this study, we used hydroxyphenyl fluorescein (HPF), which is a novel fluorescence probe with high stability against light that undergoes specific reactions with highly ROS (hROS). We examined the specificity for hROS of HPF solution and liposomal HPF in different reactions. HPF containing high cholesterol liposomes (HC-H-lipo) maintained its ability to tolerate light scattering energy, dependent on the turbidity of this solution, but DCDHF did not. In the reaction with hydroxyl radicals, the fluorescein concentration in HC-H-lipo was increased compared with that in HPF solution. In contrast, in the reaction with peroxynitrite, HC-H-lipo was inhibited. It was confirmed that HC-H-lipo that reacted specifically with hydroxyl radicals was able to distinguishably detect hydroxyl radicals. It was regarded to be useful as an examination kit. Tissue disorder induced by sodium valproate (SV) was one of the prepared murine disease models. The reactivity ratio of HC-H-lipo with serum to that of HPF solution was markedly different between the normal group (30%) and SV group (70%). In conclusion, HC-H-lipo cannot only simply and directly detect ROS but can also selectively detect ROS type. This nanocarrier is expected to be used as a novel diagnosis method.     

Two-photon absorption properties of self-assemblies of butadiyne-linked bis(imidazolylporphyrin)

Supramolecular porphyrin self-assemblies have been prepared from butadiyne-linked bis(imidazolylporphyrin) by complementary coordination of imidazole to zinc, and their two-photon absorption (2PA) and higher-order nonlinear absorption properties were investigated over femtosecond time scales using an open-aperture Z-scan method. The self-assembled porphyrin dimer of the conjugated monozinc bisporphyrin 7D was shown to have a large 2PA cross section (7.6 x 10(3) GM, where 1 GM = 10(-50) cm(4) s molecule(-1) photon(-1)) at 887 nm. By comparison of this result with that for a meso-meso-linked porphyrin array without the butadiyne connection (3.7 x 10(2) GM at 964 nm), it was demonstrated that the predominant factor in this significant enhancement of the cross section was the expansion of porphyrin-porphyrin pi-conjugation. Self-coordination and monozinc metalation were also found to be contributing factors. Furthermore, a novel self-assembled porphyrin polymer 8P consisting of a biszinc complex with a mean molecular weight of M(n) = 1.5 x 10(5) Da was shown to exhibit an extraordinarily large two-photon absorption cross section (4.4 x 10(5) GM at 873 nm). Nanosecond Z-scan experiments for 7D and 8P were also undertaken and resulted in the measurement of large effective 2PA cross sections, including the excited-state absorption (2.1 x 10(5) GM for 7D and 2.2 x 10(7) GM for 8P, respectively). Finally, three-photon absorption was observed by femtosecond Z-scan experiments at 1188 nm (7.1 x 10(-89) m(6) s(2)) and 1282 nm (1.8 x 10(-89) m(6) s(2)), an observation which is the first of its kind in porphyrin chemistry.     

Impressive europium red emission induced by two-photon excitation for biological applications

Three triazine-based europium(III) complexes were synthesized that demonstrated strong two-photon induced europium emission with a high two-photon absorption cross-section. The modified triazine ligand of complex 3 initiated over 100% enhancement of the two-photon absorption cross-section (σ(2): 320 GM) when compared with complex 1 (σ(2): 128 GM) in a solution of DMSO. Europium complex 3 is also stable in vitro, and power-dependence curves were obtained in vitro to confirm the two-photon-induced f-f emission in HeLa cells.     

mTHPC-mediated photodynamic treatment up-regulates the cytokines VEGF and IL-1alpha

Photodynamic therapy (PDT) of cancer induces oxidative stress, which intervenes in the expression of cytokines by tumor cells. The cytokines might have either a positive or a negative impact on tumor eradication. Here, we studied the expression of cytokines vascular endothelial growth factor (VEGF) and interleukin-1alpha (IL-1alpha) in the human epidermoid carcinoma A-431 cells following m-tetra(3-hydroxyphenyl)-chlorin (mTHPC)-mediated PDT in vitro and assessed the IL-1alpha effect on VEGF expression. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay revealed the enhanced production of VEGF and IL-1alpha both on mRNA and protein levels by mTHPC-loaded cells after light exposure. The silencing of IL1A by small interfering RNA resulted in decreased production of IL-1alpha and a reduced amount of VEGF. Furthermore, exogenous recombinant IL-1alpha stimulated the VEGF expression after PDT. Thus, in addition to the cytotoxic action on the A-431 cells, mTHPC-mediated PDT stimulated the production of VEGF and IL-1alpha, and IL-1alpha contributed to the VEGF overexpression. These data establish IL-1alpha as a possible target of combined cancer treatment.     

Interaction of human plasma fibrinogen with commercially pure titanium as studied with atomic force microscopy and X-ray photoelectron spectroscopy

The surface of a biomaterial interacts with the body fluid upon implantation in the human body. The biocompatibility of a material is strongly influenced by the adsorption of proteins onto the surface. Titanium is frequently used as a biomaterial for implants in orthopedics and cardiovascular devices. Understanding the biocompatibility is very important to improve implants. The surface chemistry of an implant material and its influence on the interaction with body fluid is crucial in that perspective. The main goal of this study was to investigate the conformation of human plasma fibrinogen (HPF) adsorbed on commercially pure titanium (CP Ti) on a molecular level by means of ex situ atomic force microscopy (AFM). With X-ray photoelectron spectroscopy combined with argon ion beam depth profiling, it was shown that the oxide layer present at the surface was mainly composed of TiO2, with a small percentage of Ti2O3. Ex situ AFM imaging showed the conformation of HPF on CP Ti. Single molecules and aggregates of fibrinogen were observed. The trinodular structure of single HPF molecules (two spherical D domains at the distal ends of the extended molecule and the central spherical E domain) adsorbed onto CP Ti was visualized. Aggregate formation through the connection of the D domains of the HPF molecules was observed on CP Ti. The alphaC domains of HPF were not visible on CP Ti. The ex situ AFM images indicated conformational changes of HPF upon adsorption onto CP Ti. The conformation of the adsorbed HPF molecules was different on mica and titanium. The difference in wettability between both substrates caused a larger spread of the protein on the CP Ti surface and thus resulted in a larger perturbation to the native structure of HPF as compared to mica.     

Cyano Enone-Bearing Triterpenoid Soloxolone Methyl Inhibits Epithelial-Mesenchymal Transition of Human Lung Adenocarcinoma Cells In Vitro and Metastasis of Murine Melanoma In Vivo

Introduction of α-cyano α,β-unsaturated carbonyl moiety into natural cyclic compounds markedly improves their bioactivities, including inhibitory potential against tumor growth and metastasis. Previously, we showed that cyano enone-bearing derivatives of 18βH-glycyrrhetinic (GA) and deoxycholic acids displayed marked cytotoxicity in different tumor cell lines. Moreover, GA derivative soloxolone methyl (SM) was found to induce ER stress and apoptosis in tumor cells in vitro and inhibit growth of carcinoma Krebs-2 in vivo. In this work, we studied the effects of these compounds used in non-toxic dosage on the processes associated with metastatic potential of tumor cells. Performed screening revealed SM as a hit compound, which inhibits motility of murine melanoma B16 and human lung adenocarcinoma A549 cells and significantly suppresses colony formation of A549 cells. Further study showed that SM effectively blocked transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) of A549 cells: namely, inhibited TGF-β-stimulated motility and invasion of tumor cells as well as loss of their epithelial characteristics, such as, an acquisition of spindle-like phenotype, up- and down-regulation of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin, zona occludens-1 (ZO-1)) markers, respectively. Network pharmacology analysis with subsequent verification by molecular modeling revealed that matrix metalloproteinases MMP-2/-9 and c-Jun N-terminal protein kinase 1 (JNK1) can be considered as hypothetical primary targets of SM, mediating its marked anti-EMT activity. The inhibitory effect of SM on EMT revealed in vitro was further confirmed in a metastatic model of murine B16 melanoma: SM was found to effectively block metastatic dissemination of melanoma B16 cells in vivo, increase expression of E-cadherin and suppress expression of MMP-9 in lung metastatic foci. Altogether, our data provided valuable information for a better understanding of the antitumor activity of cyano enone-bearing semisynthetic compounds and revealed SM as a promising anti-metastatic drug candidate.     

Complement activation cascade and its regulation: relevance for the response of solid tumors to photodynamic therapy

The complement system has emerged as a prominent participant in host response elicited following treatment of solid tumors by photodynamic therapy (PDT). Activity of the complement system is tightly controlled by a series of endogenous regulatory proteins. The expression of decay-accelerating factor (DAF), complement-receptor-1-related protein y (Crry), and protectin, which are the three major mouse membrane-bound complement regulatory proteins (mCRPs), was examined following treatment of murine squamous cell carcinomas SCCVII by PDT mediated by the photosensitizer Photofrin. A marked decrease was detected in the expression of all three mCRPs on cancer cells from tumors following PDT, indicating that these cells were made more vulnerable to complement attack. In order to amplify this effect, following PDT mice were injected with antibodies neutralizing either Crry, protectin, or DAF. With anti-Crry and anti-protectin this resulted in increased tumor cure rate compared to PDT alone, while the contrary was observed with PDT plus anti-DAF combination (presumably owing to additional role of DAF in T cell signaling). Further examination including other complement regulatory proteins showed that combining antitumor PDT with antibody-mediated neutralization of factor H (soluble negative complement regulator) or injection of properdin (positive complement regulator) increased the cure rates of PDT-treated tumors. The use of various agents promoting complement activity appears promising for employment as adjuvants to PDT.