Semisolid Enteral Nutrients Alter the Pharmacokinetics of Orally Administered Levetiracetam in Rats.

Enteral nutrients (ENs) affect the plasma drug concentration of orally co-administered drugs, particularly those of antiepileptic drugs, such as phenytoin and carbamazepine. However, few studies have reported the interactions of levetiracetam (LEV), an upcoming antiepileptic drug, with ENs. In this study we aimed to investigate the pharmacokinetics of LEV in 55 rats after oral co-administration of LEV with liquid or semisolid ENs. Compared with the control group, co-administration with Terumeal ® Soft significantly decreased the plasma LEV concentration at 0.5, 1, and 2 h and area under the plasma concentration-time curve from 0 to 3 h (AUC0→3h) (P < 0.01). However, the AUC0→3h of LEV remained unchanged following the administration of Terumeal ® Soft 2 h after the initial LEV administration. Moreover, co-administration with semisolid Racol® NF delayed the absorption of LEV without decreasing the AUC0→3h, whereas liquid Racol ® NF did not alter LEV pharmacokinetics. Thus, co-administration of LEV with Terumeal® Soft reduced the absorption of LEV from the gastrointestinal tract, which was prevented by administering Terumeal ® Soft 2 h after LEV administration. Semisolid Racol ® NF altered LEV pharmacokinetics without decreasing its gastrointestinal absorption. Our findings suggested that careful monitoring of the plasma LEV levels is necessary when co-administering LEV with Terumeal ® Soft, semisolid Racol ® NF, or any other semisolid ENs, to prevent the inadvertent effects of the interaction between LEV and ENs.

Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus.

Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

Atomic force microscopy dissects the hierarchy of genome architectures in eukaryote, prokaryote, and chloroplast.

Because of its applicability to biological specimens (nonconductors), a single-molecule-imaging technique, atomic force microscopy (AFM), has been particularly powerful for visualizing and analyzing complex biological processes. Comparative analyses based on AFM observation revealed that the bacterial nucleoids and human chromatin were constituted by a detergent/salt-resistant 30-40-nm fiber that turned into thicker fibers with beads of 70-80 nm diameter. AFM observations of the 14-kbp plasmid and 110-kbp F plasmid purified from Escherichia coli demonstrated that the 70-80-nm fiber did not contain a eukaryotic nucleosome-like "beads-on-a-string" structure. Chloroplast nucleoid (that lacks bacterial-type nucleoid proteins and eukaryotic histones) also exhibited the 70-80-nm structural units. Interestingly, naked DNA appeared when the nucleoids from E. coli and chloroplast were treated with RNase, whereas only 30-nm chromatin fiber was released from the human nucleus with the same treatment. These observations suggest that the 30-40-nm nucleoid fiber is formed with a help of nucleoid proteins and RNA in E. coli and chroloplast, and that the eukaryotic 30-nm chromatin fiber is formed without RNA. On the other hand, the 70-80-nm beaded structures in both E. coli and human are dependent on RNA.

Flow in a tube with an aneurysmal sac: effect of aneurysm and stent.

SUMMARY: Ruptured saccular aneurysms were severe condition for intracerebral Condition. And the cause for their rupture is not yet clear. In this study (I), since the configurations of aneurysms are considered to be a factor for the rupture of aneurysms, several shapes has been modeled using Aspect ratio AR and inclination angle of saccular aneurysms. In vitro the parametric study has been conducted on the range of Reynolds Number in human blood flow for aneurismal models of AR=2.1 and 1.3 and q=90 degrees and 70 degrees , As results, it was con firme d that there are characteristic flow patterns with Reynolds Number, And that the aneurismal configuration has effects on the shear stress and pressure losses. II) The object of this paper is to study the effects of STENTS.We made the model of aneurysm and performed in vitro study in range of Reynolds number of human blood flows using three kinds of STENTS. As results, it was confirmed that flow pattern and pressure loss changes with the kinds of STENTS. This study aims the accumulation of data to predict the hazard of aneurysmal rupture by their shapes and STENTS.

Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding.

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes.

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.

In vitro and in vivo antibacterial activity and pharmacokinetics of SC-002 and its derivative, SC-004: new oral cephalosporins.

SC-002 is a novel oral cephalosporin possessing a unique thiadiazolylethenyl moiety at the 3 position. In the present study, it was the most active against gram-positive bacteria among oral cephalosporins such as cefdinir (CFDN), cefpodoxime, cefditoren and cefaclor (CCL). It was equal to or 16 times more active than CFDN against standard and clinical strains. In particular, against clinical isolates of Morganella morganii and Haemophilus influenzae, SC-002 was 8-64 times more active than CFDN. The antibacterial activity of SC-002 against some beta-lactam-resistant strains was superior to that of CFDN. The in vivo antibacterial activity of SC-004, a pivaloyloxymethyl ester of SC-002, was 1.2-8 times more protective against systemic infections due to Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae than that of CFDN. The therapeutic effects of SC-004 on experimental respiratory tract infections caused by S. pneumoniae or H. influenzae were superior to those of CFDN and CCL. SC-004 showed higher and longer-lasting blood levels and higher urinary excretion in pharmacokinetics in mice.

Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.

The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Crystallization and preliminary crystallographic analysis of major nitroreductase from Escherichia coli.

NADPH:nitrocompound oxidoreductase from Escherichia coli, NfsA, has been crystallized in the presence of FMN by the vapor-diffusion method using polyethylene glycol 6000 as a precipitant. The crystals belonged to the triclinic space group P1 with cell dimensions, a = 52.2, b = 52.7, c = 53.3 A, alpha = 75.1, beta = 60.1, gamma = 60.5 degrees. The crystals are expected to contain two NfsA molecules per asymmetric unit. The crystals diffracted X-rays to at least 2.3 A resolution and are appropriate for structural analysis at high resolution.

4-Phenylthiazole derivatives inhibit IL-6 secretion in osteoblastic cells and suppress bone weight loss in ovariectomized mice.

A series of 4-phenylthiazole derivatives were synthesized and tested their inhibitory effect on the interleukin-6 secretion stimulated by PTH in osteoblastic cells. SCRC2941-18, 2-amino-4-(4-chlorophenyl)-5-methylthiazole, was found to be the most potent inhibitor in the derivatives. Furthermore, SCRC2941-18 significantly suppressed the bone weight loss in the ovariectomized mice, an osteoporosis model.

Purification and characterization of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase.

ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.

1.8 A crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins.

We have solved the crystal structure of FRase I, the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri, by the multiple isomorphous replacement method (MIR) at 1.8 A resolution with the conventional R factor of 0.187. The crystal structure of FRase I complexed with its competitive inhibitor, dicoumarol, has also been solved at 2.2 A resolution with the conventional R factor of 0.161. FRase I is a homodimer, having one FMN cofactor per subunit, which is situated at the interface of two subunits. The overall fold can be divided into two domains; 80% of the residues form a rigid core and the remaining, a small flexible domain. The overall core folding is similar to those of an NADPH-dependent flavin reductase of Vibrio harveyi (FRP) and the NADH oxidase of Thermus thermophilus (NOX) in spite of the very low identity in amino acid sequences (10% with FRP and 21% with NOX). 56% of alpha-carbons of FRase I core residues could be superposed onto NOX counterparts with an r.m.s. distance of 1.2 A. The remaining residues have relatively high B-values and may be essential for defining the substrate specificity. Indeed, one of them, Phe124, was found to participate in the binding of dicoumarol through stacking to one of the rings of dicoumarol. Upon binding of dicoumarol, most of the exposed re-face of the FMN cofactor is buried, which is consistent with the ping pong bi bi catalytic mechanism.

Conversion of NfsA, the major Escherichia coli nitroreductase, to a flavin reductase with an activity similar to that of Frp, a flavin reductase in Vibrio harveyi, by a single amino acid substitution.

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

Suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption in vitro and in vivo.

The suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption was examined in vitro and in vivo. This synthetic peptidyl aldehyde was found to be a potent and selective cathepsin L inhibitor in our screening for cysteine protease inhibitors. In the pit formation assay with unfractionated rat bone cells, 1.5 nM of this compound markedly inhibited parathyroid hormone-stimulated osteoclastic bone resorption. In addition, intraperitoneal administration of this peptidyl aldehyde (2.5-10 mg/kg) for 4 weeks suppressed bone weight loss dose dependently in the ovariectomized mouse, experimental model of osteoporosis. Hydroxyproline measurement of the decalcified femurs from these ovariectomized mice suggested that this compound acts as a bone resorption suppressor through the inhibition of collagen degradation.

[The use of thermography in the assessment of the rheumatoid knee--the thermographic index and the heat distribution index].

The thermographic index (TI) and the heat distribution index (HDI) have been reported to be useful in association with the assessment of disease activity in rheumatoid arthritis. We have examined the appropriate conditions on measuring both indices. It was shown that TI and HDI should be measured at 20 degrees C and at a 20-minute equilibration time. Furthermore, a comparative evaluation of TI and HDI was made. The results showed that the HDI was more sensitive and correlated better with the clinical assessment of the severity of joint swelling than TI.

Possible mechanisms of spasmolytic action of bile salts on the isolated guinea-pig gallbladder.

The spasmolytic action of bile salts on gallbladder smooth muscle could explain the alleged relief of biliary colic seen during bile acid therapy. The mechanisms of spasmolytic action of bile salts, ursodeoxycholate and deoxycholate were studied in the isolated gallbladder of guinea-pigs. The bile salts accelerated the 45Ca-efflux from the gallbladder with synchronous relaxation and inhibited the cellular 45Ca-uptake by the depolarized muscle preparation. Further, they sensitively inhibited CaCl2-induced contraction of the depolarized muscle. The tissue cyclic AMP content of the gallbladder was significantly elevated by the bile salts. Dibutyryl cyclic AMP mimicked the effects of bile salts on the Ca-efflux and the muscle relaxation, but showed no effect on the cellular Ca-uptake. From these results, it is suggested that the bile salts produce the relaxant action through accelerating Ca-efflux, which is probably coupled with the elevation of the cellular cyclic AMP level, and through suppressing the Ca-influx across the cell membrane.

Mechanisms of spasmolytic action of bile salts in depolarized guinea-pig taenia coli.

The effects of bile salts on the calcium movements and the electrical activity of the guinea-pig taenia coli were investigated and compared with those of papaverine in order to explore the mechanisms of their spasmolytic action. Four bile salts, deoxycholate, chenodeoxycholate, ursodeoxycholate and cholate, as well as papaverine, dose-dependently relaxed the depolarized taenia coli. The bile salts and papaverine caused the acceleration of 45Ca-efflux with the synchronous muscle relaxation and inhibited the cellular 45Ca-uptake by the depolarized muscle preparation. The bile salts also inhibited the increased spike frequency and the developed tension in the depolarized taenia coli. Furthermore, the dose-relaxation curves for bile salts were shifted to the right as the external calcium ion was increased. These findings suggest that the bile salts, like papaverine, may exert their spasmolytic action through accelerating the Ca-efflux and inhibiting the Ca-influx of the smooth muscle cells.