Intracisternal administration of Angiotensin II AT1 receptor antisense oligodeoxynucleotides protects against cerebral ischemia in spontaneously hypertensive rats

Pharmacological blockade of peripheral and brain Angiotensin II (Ang II) AT(1) receptors protects against brain ischemia. To clarify the protective role of brain AT(1) receptors, we examined the effects of specific antisense oligodeoxynucleotides (AS-ODN) targeted to AT(1) receptor mRNA administered intracisternally to spontaneously hypertensive rats (SHRs), 4 and 7 days before middle cerebral artery (MCA) occlusion, and we determined the infarct size and tissue swelling 24 h after surgery. A single intracisternal injection of AT(1) mRNA receptor antisense oligodeoxynucleotides reduced systemic blood pressure for 5 days and AT(1) receptor binding for at least 4 days in the area postrema and the nucleus of the solitary tract. A similar injection of scrambled oligodeoxynucleotides (SC-ODN) was without effect. Both blood pressure and AT(1) receptor binding returned to normal 7 days after antisense receptor mRNA administration. Both the infarction size and the tissue swelling after middle cerebral artery occlusion were reduced when the antisense oligodeoxynucleotide was administered 7 days, but not 4 days, before the operation. We conclude that 4 to 5 days of decrease in brain AT(1) receptor binding by a single administration of an AT(1) receptor mRNA oligodeoxynucleotide are sufficient to significantly protect the brain against ischemia resulting from total occlusion of a major cerebral vessel.     

Galactose-containing polysaccharides from Dictyostelium mucoroides as possible acceptor molecules for cell-type specific galactosyl transferase

Dictyostelium discoideum has polysaccharides that accept galactose residues by the action of cell-type-specific galactosyl transferase. This paper describes partial purification of the major galactose-accepting polysaccharide that was isolated from the related strain, Dictyostelium mucoroides and proposes its plausible carbohydrate sequences. The most potent acceptor activity was observed in the neutral and Ricinus communis agglutinin (RCA-60) bound galactosaminoglycan, consisting of galactose (Gal) and galactosamine (GalN). However, the acceptor polysaccharides are highly heterogeneous in the reactivity with RCA-120 and in molecular mass. The peak fraction was analyzed by (1)H- and (13)C-NMR studies, methylation analysis, beta-D-galactosidase digestion, controlled Smith degradation and reducing terminal analysis. Based on these results, we tentatively propose the following novel type of the common carbohydrate sequences as the acceptor polysaccharides. [abstract - see text].     

Firefly luciferase exhibits bimodal action depending on the luciferin chirality

Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas d-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.     

Hepatocyte growth factor/scatter factor suppresses TNF-alpha-induced E-selectin expression in human umbilical vein endothelial cells

Induction of E-selectin on endothelial cell surface initiates leukocyte adhesion and subsequent migration into the subendothelium. Here, we tested the effect of hepatocyte growth factor (HGF) on inflammatory cytokine-induced expression of E-selectin and consequent leukocyte-endothelial cell interaction using human umbilical vein endothelial cells (HUVEC). Prior treatment of HUVEC with HGF significantly attenuated the tumor necrosis factor (TNF)-alpha-induced E-selectin protein, adhesion of HL60 cells to HUVEC and E-selectin mRNA expression in a dose-dependent manner, while HGF itself did not exert any effects. The HGF effects on the mRNA expression were inhibited in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, which also abolished HGF-stimulated eNOS activity. These results suggest HGF plays cardiovascular protective functions mediated, at least in part, through nitric oxide-dependent suppression of inflammatory cytokine-induced E-selectin expression and subsequent tethering of leukocytes to endothelial cells.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the α subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, and , and converts chorismate to anthranilate. Two or more AS -subunit genes have been identified and characterized in several land plants. Although subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS -subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a subunit to achieve maximal enzymatic activity even with NH ₄ ⁺ as the amino donor. The V _max and K _i for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.