Cooperation of fibronectin with lysophosphatidic acid induces motility and transcellular migration of rat ascites hepatoma cells

We have previously shown that the transcellular migration of rat ascites hepatoma (AH130-MM1) cells through a cultured mesothelial cell monolayer (MCL) is triggered with lysophosphatidic acid (LPA) that stimulates actin polymerization and myosin light chain phosphorylation through the activation of Rho-ROCK (Rho-kinase) cascade. When, however, the motility of MM1 cells on a glass surface was tested by phagokinetic track motility assay, LPA failed to induce the motility. Nevertheless, when the glass had been coated with fibronectin (FN), LPA could induce phagokinetic motility which was accompanied by transformation of MM1 cells to fusiform-shape and assembly of focal adhesion. beta1 integrin, the counter receptor of FN, was expressed on MM1 cells. Anti-FN antibody, anti-beta1 integrin antibody and cyclo-GRGDSPA remarkably suppressed LPA-induced phagokinetic motility. These antibodies suppressed LPA-induced transcellular migration through MCL, as well. These results indicate that actin polymerization and phosphorylation of myosin light chain through Rho activation are insufficient for inducing motility but the cooperative FN/beta1 integrin-mediated adhesion is necessary for both the phagokinetic motility and transcellular migration of MM1 cells.     

Recombinant extracellular matrix-like proteins with repetitive elastin or collagen-like functional motifs

Using overlap elongation PCR, we created repetitive DNA libraries encoding the elastin VPGVG and collagen-like GERGDRGDP sequences. From these libraries we isolated two repetitive DNA sequences, Col-5 encoding [(GERGDRGDP)₅GER], and Ela-16 encoding [(VPGVG)₁₆VPG]. Both proteins were expressed as thioredoxin fusion proteins. The resulting recombinant extracellular matrix-like proteins had the expected properties (cell adhesive ability and thermally responsive structural change) of the functional motif sequence unit used.     

6-Amino-6-deoxy-chitosan. Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier

The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80 degrees C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan.     

Catalysis by polymer complexes. I. Enhanced esterolytic reactivity of hydroxamate–polymer complexes

N-methylmyristohydroxamic acid (1) bound to polymer micelles of laurylated poly(2- and 4-vinylpyridines) (lauryl group contet: 2VP-L, 30 mol%; 4VP-L, 33 mol%) quantitatively reacted with p-nitrophenyl acetate (NpAc) within a few seconds at 30°C, pH 8.95. Second order rate constants k_a were 34,000 M^?1 sec^?1 for 1–2VP-L and 11,400 M^?1 sec^?1 for 1–4VP-L at μ = 0.5, and they were pronouncedly improved by a decrease in ionic strength (k_a = 27,500–80,200 M^?1 sec^?1 at μ = 0.08). In contrast, poly(N-ethyl-4-vinylpyridinium bromide) hardly affected the nucleophilicity of the hydroxamate ion. Therefore, the enhancement was considered to be associated with some micellar characteristics. Typical saturation phenomena of the reaction rate were observed for p-nitrophenyl hexanoate (NpOCOPe) and 3-nitro-4-acetoxybenzoic acid (NpAcCOOH). It was suggested that binding of NpOCOPe is caused by the hydrophobic interaction, while that of NpAcCOOH is probably induced by the electrostatic interaction. It is demonstrated that the cationic polymer micelle enormously activates the bound hydroxamate anion, and these complexes would be of much interest as a biomimetic system for enzyme catalysis.     

Marine Heterotrophic Amoebae, Flagellates and Heliozoa From Belize (Central America) and Tenerife (Canary Islands), With Descriptions of New Species, Luffisphaera Bulbochaete N. Sp., L. Longihastis N. Sp., L. Turriformis N. Sp. and Paulinella Intermedia N. Sp.

ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids, the bicosoecids, the chromulinids, the pedinellids and a variety of laxa of uncertain affinities (Protista incertae sedis). the identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described: one new species of the amoeba genus Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Luffisphaera Belcher & Swale, 1975 ( Luffisphaera bulbochaete n. sp.; L. longihastis n. sp.; L. turriformis n. sp.).     

New Thioureas and Related Substances Intended for Melanoma Targeting

Thiouracil and a few related drugs are known to be melanoma-seeking agents owing to specific incorporation into nascent melanin. The melanin-affinic properties are apparently due to binding to intermediates, preferably dopaquinone, produced in the melanin synthetic pathway by tyrosinase-catalysed oxidation of tyrosine. In the present paper, in vitro screening methods have been used for the identification of possible melanoma seekers according to the above principle. The binding of test substance to dopaquinone suppresses dopachrome formation by the withdrawal of dopaquinone from the reaction mixture, and the decrease in dopachrome concentration was monitored spectrophotometrically at 475 nm. In order to eliminate false results caused by tyrosinase inhibition, which also will decrease the dopachrome concentration, the oxygen consumption was followed potentiometrically. To avoid the effect of tyrosinase inhibition on dopachrome formation, additional experiments with autoxidation of L-dopa in the presence of test substance were performed. Of the 22 substances (mainly thioureylenes and thioamides) studied, 4,5,6-triamino-2(H)-pyrimidinehtionsulfate, trithiocyanuric acid, 2-thiouracil, 6-methyl-2-thiouracil, and 4-amino-2-mercaptopyrimidine most effectively decreased the dopachrome formation with no or little inhibition of tyrosinase activity. They should therefore be regarded as potential melanoma seekers. In a complementary autoradiographic study on the uptake of the potent tyrosinase inhibitor mercaptobenzothiazole (MBT) in B 16 melanoma, transplanted to mice, it was found that strong tyrosinase inhibition seems to decrease incorporation into melanin in vivo. MBT was partly accumulated in restricted areas of the tumor, which may be explained by the small molar dose injected.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.