Hybrid liposomes inhibit the growth of Cholangiocarcinoma by induction of cell cycle arrest in G1 phase

Specific accumulation and cell cycle arrest were observed in human cholangiocarcinoma cells by hybrid liposomes composed of 90 mol % l-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol % polyoxyethylene(21)dodecyl ether (C₁₂(EO)₂₁) without affecting normal cholangiocytes.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Enhancement of neurite outgrowth-promoting activity by heparin derivatives in sodium chlorate-treated explant cultures of rat central neurons

Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH. Digestion of OR2DSH with heparitinases generated unsaturated disaccharides, comprising 86.5% DeltaDiHS-(6,N)S (DeltaUA1-->4GlcNS(6S)) and 13.5% DeltaDiHS-NS (DeltaUA1-->4GlcNS), as well as undigested oligosaccharides in which uronate moieties were derivatized by the cleavage of the covalent bond between the C-2 and C-3 positions by periodate-oxidation. The molecular mass of OR2DSH was determined to be 11 kDa, which is almost the same as those of other heparin derivatives such as 2-O-desulfated heparin (2DSH), 6-O-desulfated heparin (6DSH) and N-desulfated N-reacetylated heparin (NDSNAc-heparin). The ability of OR2DSH to enhance neurite outgrowth-promoting activity was evaluated using the explant culture of neocortical tissue from rat embryo in which endogenous heparan sulfate at the cell surface lost substantial numbers of sulfate groups by the action of 40 micro M sodium chlorate. The maximum activity of OR2DSH (29.7%) was achieved at 10 micro g/ml, and those of OR-heparin (21.7%), 2DSH (18.7%) and intact heparin (16.3%) were 100 micro g/ml, whereas that of NDSNAc-heparin (16.5%) was 1,000 micro g/ml. Completely 6-O-desulfated heparin (100:6DSH) exhibited very weak activity (3.3%) at 1,000 micro g/ml. These results suggest that the potency of OR2DSH to enhance neurite outgrowth-promoting activity is exerted synergetically by two different components in OR2DSH, i.e., the IdoA alpha1-->4GlcNS(6S) unit, which contains 6-O- and 2-N-sulfate groups, and the uronate moiety in which the covalent bond between C-2 and C-3 is cleaved, although the mode of action remains to be clarified.     

Nucleotide sequence of phospholipase A(2) gene expressed in snake pancreas reveals the molecular evolution of toxic phospholipase A(2) genes

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.     

Laminin-6 is activated by proteolytic processing and regulates cellular adhesion and migration differently from laminin-5

Laminin-6 (LN6) and laminin-5 (LN5), which share the common integrin-binding domain in the laminin alpha3 chain, are thought to cooperatively regulate cellular functions, but the former has poorly been characterized. Human fibrosarcoma HT1080 cells expressing an exogenous alpha3 chain were found to secrete LN6 with the full-length alpha3 chain and a smaller amount of its processed form lacking the carboxyl-terminal G4-5 domain, besides mature LN5 without G4-5 (mat-LN5). We prepared the unprocessed LN6 and mat-LN5, as well as LN6 mutants without G4-5 (LN6DeltaG4-5) or G5 (LN6DeltaG5). These laminins supported attachment of HT1080 cells and human keratinocytes (HaCaT) through integrins alpha(3)beta(1) and/or alpha(6)beta(1). LN6DeltaG4-5, LN6DeltaG5, and mat-LN5 promoted rapid cell spreading, whereas LN6 did hardly. A purified G4-5 fragment of the laminin alpha3 chain supported cell attachment through interaction with heparan sulfate proteoglycans and promoted cell spreading in combination with mat-LN5 or LN6DeltaG4-5. These results imply that the G4-5 domain within the LN6 molecule suppresses cell adhesion, while the released G4-5 promotes it. The presence of G5 rather than the heparin-binding domain G4 was responsible for the impaired cell spreading activity of LN6. However, the unprocessed LN6 promoted cell spreading in the presence of mat-LN5. Unlike mat-LN5, both LN6DeltaG4-5 and LN6 did weakly or did not stimulate cell motility. These findings demonstrate that LN6 and LN5 have distinct biological activities, but they may cooperatively support cell adhesion. The proteolytic processing of the alpha3 chain seems to regulate the physiological functions of LN6.     

Modification of di- and tetrasaccharides from shark cartilage keratan sulphate by refined anhydromethanolic hydrochloric acid-treatments and evaluation of their specific desulphation

Highly sulphated keratan di- and tetrasaccharides were prepared from keratan sulphate (KS) of shark cartilage by enzymatic digestion with keratanase II and subsequent chromatography. The tetrasaccharide fraction carrying four sulphate groups was completely desulphated by 100 mM anhydromethanolic hydrochloric acid (MeOH-HCl) treatment at room temperature for 16 h. The conditions for the desulphation reaction by MeOH-HCl treatment were examined using sulphated keratan di- and tetrasaccharides as substrates by means of reversed phase high performance liquid chromatography (HPLC) and/or capillary electrophoresis, followed by the preparation of partially desulphated keratan oligosaccharides. Sulphate substitution patterns of monosulphated keratan disaccharide and trisulphated keratan tetrasaccharide were evaluated by methylation analysis. The results suggested that 6-O-sulphate groups of Gal moieties are cleaved faster than those of GlcNAc moieties under the present conditions adopted for the MeOH-HCl treatment of KS-derived oligosaccharides.     

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of alpha3 chain

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.     

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.     

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.     

The Traf2- and Nck-interacting kinase as a putative effector of Rap2 to regulate actin cytoskeleton

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific roles in cell signaling remain unknown. In the present study, we have affinity-purified from rat brain a Rap2-interacting protein of approximately 155 kDa, p155. By liquid chromatography tandem mass spectrometry, we have identified p155 as Traf2- and Nck-interacting kinase (TNIK). TNIK possesses an N-terminal kinase domain homologous to STE20, the Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase kinase, and a C-terminal regulatory domain termed the citron homology (CNH) domain. TNIK induces disruption of F-actin structure, thereby inhibiting cell spreading. In addition, TNIK specifically activates the c-Jun N-terminal kinase (JNK) pathway. Among our observations, TNIK interacted with Rap2 through its CNH domain but did not interact with Rap1 or Ras. TNIK interaction with Rap2 was dependent on the intact effector region and GTP-bound configuration of Rap2. When co-expressed in cultured cells, TNIK colocalized with Rap2, while a mutant TNIK lacking the CNH domain did not. Rap2 potently enhanced the inhibitory function of TNIK against cell spreading, but this was not observed for the mutant TNIK lacking the CNH domain. Rap2 did not significantly enhance TNIK-induced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergent-insoluble cytoskeletal fraction. These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.