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Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Let G be a graph in which each vertex v has a positive integer weight b ( v ) and each edge ( v , w ) has a nonnegative integer weight b ( v , w ) . A bandwidth consecutive multicoloring, simply called a b-coloring of G, assigns each vertex v a specified number b ( v ) of consecutive positive integers as colors of v so that, for each edge ( v , w ) , all integers assigned to vertex v differ from all integers assigned to vertex w by more than b ( v , w ) . The maximum integer assigned to vertices is called the span of the coloring. The b-coloring problem asks to find a b-coloring of a given graph G with the minimum span. In the paper, we present four efficient approximation algorithms for the problem, which have theoretical performance guarantees for the computation time, the span of a found b-coloring and the approximation ratio. We also obtain several upper bounds on the minimum span, expressed in terms of the maximum b-degrees, one of which is an extension of Brooks' theorem on an ordinary coloring.

Article, TETRAHEDRON LETTERS. 49(3): 552-556 (2008)

Let G be a graph with a positive integer weight ω(v) for each vertex v. One wishes to assign each edge e of G a positive integer f(e) as a color so that ω(v) ≤ |f(e) − f(e′)| for any vertex v and any two edges e and e′ incident to v. Such an assignment f is called an ω-edge-coloring of G, and the maximum integer assigned to edges is called the span of f. The ω-chromatic index of G is the minimum span over all ω-edge-colorings of G. In the paper, we present various upper and lower bounds on the ω-chromatic index, and obtain three efficient algorithms to find an ω-edge-coloring of a given graph. One of them finds an ω-edge-coloring with span smaller than twice the ω-chromatic index.

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease. It has been generally accepted that the proapoptotic property of the familial ALS (FALS)-linked mutant SOD1 genes plays an important role in the pathogenesis of some FALS cases. We found here that expression of N19S-SOD1, a novel SOD1 mutant originally found in a sporadic ALS patient, induces lower grade death in NSC34 cells than FALS-linked mutant SOD1. In agreement, intracytoplasmic aggregate formation and SOD1 polymerization are less prominently induced by ectopic expression of N19S-SOD1 than FALS-linked mutant SOD1. We further found that additional cell stresses, such as inhibition of proteasomal activity or up-regulation of intracellular oxidative stress, enhance N19S-SOD1-induced aggregate formation and polymerization of N19S-SOD1. Such analysis of the intracellular polymerization and the ubiquitination of N19S-SOD1 have further suggested that it is recognized as a misfolded protein, like FALS-linked mutant SOD1, whereas wild-type SOD1 is not. Altogether, it is speculated that the N19S mutation of SOD1 in cooperation with associated cell stresses contributes to the onset of ALS as a risk factor.

Let G be a graph in which each vertex v has a positive integer weight b(v) and each edge (v,w) has a nonnegative integer weight b(v,w). A bandwidth consecutive multicoloring, simply called a b-coloring of G, assigns each vertex v a specified number b(v) of consecutive positive integers as colors of v so that, for each edge (v,w), all integers assigned to vertex v differ from all integers assigned to vertex w by more than b(v,w). The maximum integer assigned to vertices is called the span of the coloring. The b-coloring problem asks to find a b-coloring of a given graph G with the minimum span. In the paper, we present four efficient approximation algorithms for the problem, which have theoretical performance guarantees for the computation time, the span of a found b-coloring and the approximation ratio. We also obtain several upper bounds on the minimum span, expressed in terms of the maximum b-degrees, one of which is an extension of Brooks’ theorem on an ordinary coloring.

cDNA generated from lipopolysaccharide-stimulated bovine peripheral blood mononuclear cells was used to amplify and clone the bovine interleukin-1 receptor antagonist (IL-1ra) using primers derived from semi-conserved regions between human and mouse IL-1ra sequences. 5' and 3' terminal sequences of bovine IL-1ra were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of bovine IL-1ra demonstrated 80%, 78%, 78%, 77% and 76% homology with human, mouse, rat, rabbit and equine sequences, respectively. Recombinant bovine IL-1ra produced in Escherichia coli suppressed the growth inhibitory activity of bovine IL-1beta on A375 cells in a dose-dependent manner, indicating that the present bovine IL-1ra cDNA encodes biologically active proteins.

TNF-alpha has been reported to be induced in mice infected with Sendai virus. We evaluated the role of TNF-alpha in the virus infection. TNF-alpha was induced locally in proportion to virus titers in the lung. The activity was correlated with suppression of body weight gain. Passive immunization against TNF-alpha improved body weight gain and ameliorated pneumonic lesions in infected mice, and prevented them from lethal infection, but lung virus induced emaciation, pneumonic lesions and death were mediated by TNF-alpha.

The effects of the new anti-platelet agent KBT-3022, ethyl 2-[4,5-bis(4-methoxyphenyl)-thiazol-2-yl]pyrrol-1-ylacetate, and its metabolite desethyl KBT-3022 on rabbit neutrophil function were investigated in comparison with the effects of acetylsalicylic acid (ASA), ticlopidine hydrochloride (TP), cilostazol (CIL) and indomethacin (IM). The adhesion and migration of neutrophils induced by formyl-methionyl-leucyl-phenylalanine (fMLP) were inhibited by all the compounds tested, their rank order of potency being KBT-3022 = desethyl KBT-3022 > TP = CIL = IM > ASA. KBT-3022, desethyl KBT-3022, CIL and IM all suppressed fMLP-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophils, their potencies correlating with their inhibitory effects on fMLP-induced adhesion and migration. KBT-3022 (1 microM), desethyl KBT-3022 (1-10 microM) and CIL (10 microM) but not IM significantly inhibited both neutrophil migration and the increase in [Ca2+]i induced by leukotriene B4 (LTB4). KBT-3022 (1 microM) and desethyl KBT-3022 (1 microM) suppressed the increase in [Ca2+]i induced by complement C5a. Although KBT-3022 and desethyl KBT-3022 did not influence [3H]LTB4 and [125I]C5a specific binding, [3H]fMLP specific binding was inhibited by desethyl KBT-3022 (IC50: 1.9 microM). Neutrophil adhesion and superoxide anion production stimulated by phorbol 12-myristate 13-acetate were partially inhibited by KBT-3022 (1 microM) and desethyl KBT-3022 (1-10 microM). These results suggest that KBT-3022 and desethyl KBT-3022 have a wider spectrum of action and are more potent inhibitors of neutrophil activation than ASA, TP, CIL and IM.

Let [Formula: see text] be a graph with a positive integer weight [Formula: see text] for each vertex [Formula: see text]. One wishes to assign each edge [Formula: see text] of [Formula: see text] a positive integer [Formula: see text] as a color so that [Formula: see text] for any vertex [Formula: see text] and any two edges [Formula: see text] and [Formula: see text] incident to [Formula: see text]. Such an assignment [Formula: see text] is called an [Formula: see text]-edge-coloring of [Formula: see text], and the maximum integer assigned to edges is called the span of [Formula: see text]. The [Formula: see text]-chromatic index of [Formula: see text] is the minimum span over all [Formula: see text]-edge-colorings of [Formula: see text]. In the paper, we present various upper and lower bounds on the [Formula: see text]-chromatic index, and obtain three efficient algorithms to find an [Formula: see text]-edge-coloring of a given graph. One of them finds an [Formula: see text]-edge-coloring with span smaller than twice the [Formula: see text]-chromatic index.

In order to obtain basic information about bovine interleukin-1 (IL-1 beta), levels of IL-1 beta in sera and milk of clinically normal mature Holstein cattle before and after parturition and in sera of newborn calves were examined by ELISA. The level of IL-1 beta was undetectable in sera of mature cattle around the time of artificial insemination, but the concentration gradually increased and reached a peak at parturition and then decreased again to an undetectable level. IL-1 beta in milk was detected on the day of parturition but not thereafter. IL-1 beta mRNA was detected by reverse transcription-polymerase chain reaction in the cells from milk collected during 20 days before and 2 to 3 days after parturition, but was not detected thereafter. Although IL-1 beta was not detected in all the sera of newborn calves, the concentration transiently increased with peak titers on day 3 and became undetectable by day 14 after birth. Newborns that showed serum IL-1 beta on day 3 had been fed on colostrum in which the IL-1 beta concentration was significantly higher than that in colostrum that had been fed to newborns having no detectable IL-1 beta on day 3. These results indicate that IL-1 beta is induced in association with pregnancy in healthy dairy cattle and that the cytokine might be transferred to neonates via colostrum.

The synthesis and biological evaluation of orally active inhibitors of matrix metalloproteinase are reported. Modifications of the P2' position and the alpha-substituent of hydroxamic acid derivatives were carried out, and revealed that the P2' substituent influenced the MMP inhibitory activities in vitro and in plasma after oral administration. The hydroxamates with phenylglycine at the P2' position were absorbed well orally. Compound 15e, which exhibited the longest duration of inhibitory activity in plasma after oral administration among the phenylglycine derivatives (5a-5d, 15a, 15c, 15e), was evaluated in a rat adjuvant arthritis model. A reduction in hind foot pad swelling and improvements of some inflammatory parameters were demonstrated when the compound was administered orally. These results indicate the potential of MMP inhibitors for rheumatoid arthritis.

We devised a method for evaluating polymorphonuclear leukocyte (PMN) infiltration in vivo employing an air bleb technique combined with measurement of myeloperoxidase (MPO) activity, and the effects of some anti-platelet agents were evaluated. KBT-3022 (ethyl 2-[4,5-bis (4-methoxyphenyl)thiazol-2-yl] pyrrol-1-ylacet ate) and cilostazol inhibited the increase in MPO activity in the connective tissue around the air bleb induced by leukotriene B4 (LTB4) and formyl-methionyl-leucyl-phenylalanine (fMLP). Indomethacin inhibited only the fMLP-induced increase in MPO activity, but ticlopidine hydrochloride and acetylsalicylic acid had no effect. Histologic observation confirmed the inhibition of PMN infiltration by KBT-3022. These results indicate that KBT-3022 may be a potent inhibitor of both LTB4- and fMLP-induced infiltration of PMNs.

The 55- (H-UK) and 36-kDa forms (L-UK) of human urinary urokinase lost most of esterase activity toward acetyl-glycyl-L-lysine methyl ester upon reductive cleavage of 3 SS bonds with dithiothreitol in the presence of the competitive inhibitor, Nα-benzoyl-L-arginine amide (BAA), bound to polyacrylyl azide with C 16 N 3 -arm (PAA) at 0.3 M guanidine, a threshold point of the native state where a protein-denaturating transition began. One of the 3 SS bonds was protected from reduction, with an unaltered activity, under the similar conditions except for replacement of BAA-PAA conjugate by glycine-PAA conjugate. This “specific” SS bond was reduced and, after the other SH groups produced were blocked with iodoacetamide (IAM), selectively reoxidized, which resulted in complete reactivation. The intact B-chain isolated from H-UK was completely inactivated when its specific SS bond was reduced and selectively alkylated with IAM after the other SH groups were reversibly blocked with 5, 5′-dithiobis(2-nitrobenzoic acid), which was finally removed. The results indicate that a single specific SS bond is essential for retaining a conformation necessary to activity exhibition.

pH-stability curves and heat inactivation of the MW 55000 form (H-UK) and MW 36000 form (L-UK) of human urinary urokinase [EC 3. 4. 21. 31] were investigated by measurements of plasminogen activator activity. L-UK was distinctly more stable than H-UK (in terms of activity) on incubation at pH 6.8-9.6 or on heat treatment at temperatures above 60°. The ranges of pH 8.2-9.0 and 5-37°, were favorable for both H-UK and L-UK.