Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides.

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by alpha-methyl-gamma,gamma-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(alpha-methyl-gamma,gamma-diphenylallenecarbonyl)-L-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pK(a) value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.

Antiinflammatory activity of heat-treated Cassia alata leaf extract and its flavonoid glycoside.

Antiinflammatory activities of heat-treated Cassia alata leaf extract and kaempferol 3-O-gentiobioside (K3G) isolated from C. alata as an abundant flavonoid glycoside were studied by comparing their activities with the activities of sun-dried C. alata leaf extract. We observed strong inhibitory effects on Concanavalin A-induced histamine release from rat peritoneal exudate cells both in the extracts of heat-treated and sun-dried C. alata leaves. Furthermore, the heat-treated leaf extract exhibited stronger inhibitory effects than the effects of the sun-dried leaf extract at low concentrations in the studies of Concanavalin A-induced histamine release, 5-lipoxygenase inhibition, and also inhibition of cyclooxygenases (COX-1 and COX-2), whereas K3G showed weak inhibitory effects on Concanavalin A-induced histamine release, 5-lipoxygenase, and COX-1. No anti-hyaluronidase effect was detected in any of the materials tested.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2002].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 899 strains of Gram-positive bacteria, 1500 strains of Gram-negative bacteria, and 158 strains of anaerobic bacteria obtained from 28 medical institutions during 2002 was measured. The results were as follows; 1. MEPM was more active than other carbapenem antibiotics against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MIC90 of MEPM against Pseudomonas aeruginosa was the lowest of the drugs tested. MEPM showed low cross-resistant rate against both imipenem-resistant P. aeruginosa and ciprofloxacin-resistant P. aeruginosa. MEPM was active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE). 2. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (4 strains) in Escherichia coli and 1.9% (2 strains) in Klebsiella pneumoniae. Carbapenems including MEPM were active against these ESBL strains. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem; at present, 7 years after available for commercial use.

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans.

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.

Salt stress-induced dissociation from cells of a germin-like protein with Mn-SOD activity and an increase in its mRNA in a moss, Barbula unguiculata.

To estimate the physiological roles of a germin-like protein (BuGLP) with Mn-SOD activity isolated newly from a moss, Barbula unguiculata, BuGLP mRNA levels during cell growth and the effects of methyl viologen and salt stress were studied. BuGLP mRNA levels were at their peak during the exponential phase of growth and decreased thereafter, but SOD activity was held at the same level as that during the exponential phase. When methyl viologen was present as a generator of superoxide the amount of BuGLP transcripts decreased, but that of SOD activity of BuGLP bound to the cell wall was not affected. The addition of NaCl to the cells during the logarithmic phase increased both the BuGLP mRNA levels and total SOD activity of BuGLP, but decreased the SOD activity bound to the cell wall due to release of most of the SOD activity into the medium. On the other hand, the addition of NaCl to the cells during the stationary phase hardly affected BuGLP mRNA levels or SOD activity levels bound to the cell wall. These results suggest that the induction of BuGLP gene by salt stress is caused by dissociation of BuGLP protein from the cell wall into the medium in the cells during the logarithmic phase.

Molecular cloning and expression of HRLRRP, a novel heart-restricted leucine-rich repeat protein.

We isolated a novel leucine-rich repeat protein (LRRP) cDNA from E13 mouse embryos by the in silico approach. The cDNA encoded a protein of 274 amino acids having 7 leucine-rich repeat motifs at the center of the protein. An in vitro transcription/translation study showed that the cDNA coded for a peptide of approximately 31kDa. Northern blot analysis suggested that the mRNA of this novel LRRP was expressed only in the heart, although RT-PCR indicated slight expression in skeletal muscle as well. The transcripts of this gene and Nkx-2.5/Csx were detected in the early stage of cardiac differentiation of P19CL6 embryonal carcinoma cells treated with 1% dimethyl sulfoxide. The fusion protein made between it and GFP was detected at a high level in mitochondria and a low level in the nuclei of COS7 cells. The nuclei of the adult mouse heart were strongly stained with the antibody raised against the synthetic peptide of the protein. Therefore, we designated the gene as heart-restricted leucine-rich repeat protein (HRLRRP) and assume that mouse HRLRRP may play important roles in cardiac development and/or cardiac function.

An abundant periplasmic protein of the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans is PstS, a component of an ABC phosphate transport system.

To understand a physiological role of an abundant 34-kDa periplasmic protein in the denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans grown in a medium containing malate as the carbon source, the gene for the protein was isolated. The deduced amino acid sequence of the protein had a sequence similarity of 66.2% to that of PstS from Sinorhizobium meliloti. The downstream sequence of the Rhodobacter pstS contained five genes similar to pstCAB and phoUB, and its upstream sequence contained a putative regulatory sequence that is analogous to the Pho box involved in phosphate-limitation-induced gene expression in Escherichia coli. Both the amount of the PstS and the pstS promoter-driven expression of lacZ activity increased about two-fold in response to phosphate limitation. This is the first isolation of pst genes encoding proteins of an ABC phosphate transporter system from phototrophic bacteria.

Germin-like protein gene family of a moss, Physcomitrella patens, phylogenetically falls into two characteristic new clades.

We identified 77 EST clones encoding germin-like proteins (GLPs) from a moss, Physcomitrella patens in a database search. These Physcomitrella GLPs ( PpGLP s) were separated into seven groups based on DNA sequence homology. Phylogenetic analysis showed that these groups were divided into two novel clades clearly distinguishable from higher plant germins and GLPs, named bryophyte subfamilies 1 and 2. PpGLPs belonging to bryophyte subfamilies 1 lacked two cysteines at the conserved positions observed in higher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2 contained two cysteines as observed in higher plant germins and GLPs. In bryophyte subfamily 1, 12 amino acids, in which one of two cysteines is included, were deleted between boxes A and B. Further, we determined the genomic structure of all of seven PpGLP genes. The sequences of PpGLP s of bryophyte subfamily 1 contained one or two introns, whereas those of bryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, a liverwort GLP from Marchantia polymorpha , and two moss GLPs from Barbula unguiculata and Ceratodon purpureus also fell into bryophyte subfamily 1 and bryophyte subfamily 2, respectively. No higher plant germins and GLPs were grouped into the bryophyte subfamilies 1 and 2 by our analysis. Moreover, we revealed that PpGLP6 had manganese-containing extracellular superoxide dismutase activity. These results indicated that bryophyte possess characteristic GLPs, which phylogenetically are clearly distinguishable from higher plant GLPs.

Synthesis and biological activities of fluorinated chalcone derivatives.

We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe(3+)-ADP induced NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-2',4'-dimethoxy chalcone (7) was the most effective compound in the in vitro assay using a human cancer cell line panel (HCC panel) consisting of 39 systems.