Adhesion behavior of peritoneal cells on the surface of self-assembled triblock copolymer hydrogels

Adhesion behavior of cells to the surface of physical hydrogel membranes prepared by water-induced self-organization of precisely synthesized ABA-triblock copolymers comprised of poly(beta-benzyl L-aspartate) (PBLA) as A segment and poly(ethylene oxide) (PEO, molecular weight = 20 000) as the B segment were investigated. The cast film from the methylenechloride solution of these copolymers swelled in water very rapidly forming hydrogels (100-400% water content of total weight). The content of PBLA affected the strength, the hydrophobicity, and the amount of water involved in the hydrogel surface. During the early stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18 (20K18) and 25 (20K25) monomeric units was not observed, while adhesion on the hydrogels of PEO and PBLA with 32 (20K32) and 55 (20K55) monomeric units was successful, suggesting more than 12 mol % in PBLA content is necessary for adhesion of these cells. Although cell spreading on the hydrogels of 20K18, 20K25, and 20K32 was not sufficient, the hydrogel of 20K55 allowed cell adhesion and spreading to be bipolar with leading edge whose raffling is active with pseudopodium and lamellipodium as well as PBLA homopolymer, suggesting active motility of these cells. Remarkably, prolonged incubation restored adhesiveness onto the films at 20K18 in contrast to adhesion with 20K25 despite low hydrophobicity. It is conceivable that adaptation of proteins and chemical changes to the surface during the culture period may participate in these phenomena. Mechanical properties and interaction between cell and these copolymer hydrogels could be controlled by composition of block segments, and optimization for implants could also be attainable.     

Piericidins C5 and C6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.     

Targeted disruption of intracellular type I platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis

Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits alpha1 and alpha2 as well as LIS1, a product of the causative gene for type I lissencephaly. LIS1 plays an important role in neuronal migration during brain development, but the in vivo function of the catalytic subunits remains unclear. In this study, we generated alpha1- and a2-deficient mice by targeted disruption. alpha1(-/-) mice are indistinguishable from wild-type mice, whereas alpha2(-/-) male mice show a significant reduction in testis size. Double-mutant male mice are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the spermatocyte stage and an increase of apoptotic cells in the seminiferous tubules. The catalytic subunits are expressed at high levels in testis as well as brain in mice. In wild-type mice, alpha2 is expressed in all seminiferous tubule cell types, whereas alpha1 is expressed only in the spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells at an early spermatogenic stage. We also found that the LIS1 protein levels, but not the mRNA levels, were significantly reduced in alpha2(-/-) and double-mutant mice, suggesting that the catalytic subunits, especially alpha2, are a determinant of LIS1 expression level.     

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.     

Crystal structure of rice alpha-galactosidase complexed with D-galactose

alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes.     

Potential changes with gamma-band oscillation at the frontal scalp elicited by intravenous olfactory stimulation in humans

Intravenous olfaction is a unique stimulation method often used in Japan to diagnose olfactory disturbances. Odorant is injected into a vein and transported by blood flow and respiration to the upper air tract. The intravenous olfaction might allow the potential at the frontal scalp to be recorded without contamination from electromyograms, such as those caused by sniffing. We injected Alinamin (thiamine propyldisulphide) into healthy subjects according to a standard protocol for clinical intravenous olfaction testing and we simultaneously recorded potential changes at the frontal scalp. When Alinamin was injected into the right median cubital vein over a 20 s period, the potential changes with gamma-band oscillations were detected 17.6 +/- 6.7 s (mean +/- SD) after the start of the injection. The main frequency component of this gamma-band oscillation is 30-160 Hz. The gamma-band oscillation elicited by intravenous olfactory stimulation (VOP) was similar to the induced wave of the olfactory bulb. Mapping the VOPs on the frontal scalp of a subject with less developed frontal sinuses and the relation between the thickness of the frontal sinuses and VOP amplitude suggest an intracranial source, possibly the olfactory bulb. The gamma-band potential at the frontal scalp is a useful measure of central disturbance.     

Radiation-induced chain reactions in alcohol solutions of diphenyliodonium salts: a high-sensitivity chemical dosimeter

Chain reactions in gamma-irradiated 2-propanol solutions of diphenyliodonium salts have been studied. Protonic acids were generated in the irradiated solutions with high yields, whereas acid formation as a result of thermal reactions was negligible. The solution can be used as a high-sensitivity chemical dosimeter. The G value of acidic protons increases with increasing concentration of diphenyliodonium salt at the lower concentrations because the reaction rate of a propagation reaction increases. However, the chain is limited by a termination reaction between phenyl radical and the iodonium salt: The G value shows a maximum value of 610 micromol J(-1) at the concentration of 0.08 mol dm(-3) and decreases at higher salt concentrations.     

The antisense approach in amyloid light chain amyloidosis: identification of monoclonal Ig and inhibition of its production by antisense oligonucleotides in in vitro and in vivo models

Primary amyloid L chain (AL) amyloidosis is a plasma cell disorder in which depositions of AL cause progressive organ failure. The lack of effective therapies for this fatal disease prompts exploration of newer treatment avenues. We have investigated the application of antisense oligonucleotides (AS) for the inhibition of monoclonal Ig production. The monoclonal L chain was identified by using primers designed for amplifying the human lambda Ig V (Vlambda) region. We demonstrated that AS against L chain complementarity-determining regions inhibited the production of L chain in vitro. RPMI 8226 myeloma cells injected in SCID mice developed s.c. tumors. RT-PCR analysis showed Vlambda mRNA expression in the tumors. In addition, the presence of human Ig in the sera of mice given injection of RPMI 8226 cells was confirmed by ELISA. Administration of AS inhibited the expression of Vlambda mRNA in the s.c. tumors and decreased the concentration of L chain in serum. Therefore, we have shown that it is possible to determine the sequence of Vlambda mRNA and design specific complementary oligonucleotides, suggesting that treatment with Vlambda antisense could represent a rational novel approach to improve treatment outcome in AL amyloidosis.