Anti-angiogenic activity of tocotrienol

The anti-angiogenic property of vitamin E compounds, with particular emphasis on tocotrienol, has been investigated in vitro. Tocotrienol, but not tocopherol, inhibited both the proliferation and tube formation of bovine aortic endothelial cells, with delta-tocotrienol appearing the highest activity. Also, delta-tocotrienol reduced the vascular endothelial growth factor-stimulated tube formation by human umbilical vein endothelial cells. Our findings suggest that tocotrienol has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.     

Structure-activity relationship study of taxoids for their ability to activate murine macrophages as well as inhibit the growth of macrophage-like cells

A series of new taxoids modified at the C-3', C-3'N, C-10, C-2 and C-7 positions has been designed, synthesized and evaluated for their potency to induce NO and TNF production by peritoneal murine macrophages (Mphi) from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ strains and human blood cells, and for their ability to inhibit the growth of Mphi-like cell lines J774.1 and J7.DEF3. The SAR-study has shown that the nature of the substituents at these positions have critical effect on the induction of TNF and NO production by Mphi. Positions C-3' and C-10 are the most flexible and an intriguing effect of the length of the substituents at the C-10 position is observed for taxoids bearing a straight chain alkanoyl moiety. An aromatic group at the C-3'N and C-2 positions is required for the activity, while only hydroxyl or acetyl substituents seem to be tolerated at the C-7 position. The natural stereochemistry in the C-13 isoserine side chain of the taxoids is an absolute requirement for macrophage activation. It has also been clearly shown that there is no correlation between the ability of the taxoids to induce TNF/NO production in C3H/HeN Mphi and the cytotoxicity against Mphi-like cells.     

Fasting increases gene expressions of uncoupling proteins and peroxisome proliferator-activated receptor-gamma in brown adipose tissue of ventromedial hypothalamus-lesioned rats

Uncoupling proteins (UCPs) are supposed to be involved in diet-induced thermogenesis. Their activities are usually elevated by feeding and reduced by fasting in normal animals. To investigate whether fasting affects the expression of UCPs mRNA in brown adipose tissue (BAT) of bilateral ventromedial hypothalamus (VMH)-lesioned rats, we determined the gene expression of UCP1, UCP2 or UCP3 in BAT of VMH-lesioned rats and examined oxygen consumption in these rats under fed or 48-h fasted conditions. Northern blotting revealed no difference in the expression of UCPs mRNA in BAT between VMH-lesioned and sham-operated rats under the fed condition, however, expressions were increased markedly in BAT of VMH-lesioned rats under the fasted condition. Under the fed condition, no difference in oxygen consumption was observed between VMH-lesioned and sham-operated rats. Under the fasted condition, oxygen consumption decreased in both rats, however, it decreased in VMH-lesioned less than in sham operated rats. To explore the mechanism that fasting elevated BAT UCPs mRNA in VMH-lesioned rats, we measured peroxisome proliferator-activated receptor (PPAR)-gamma mRNA and protein in BAT, because PPAR-gamma agonist can elevate UCPs mRNA levels in BAT. Under the fed condition, no differences in the expression of PPAR-gamma mRNA and protein content were observed between in BAT of VMH-lesioned and sham-operated rats. Under the fasted condition, however, both increased in BAT of VMH-lesioned rats. These results suggest that VMH-lesions enhance the gene expression of UCPs in BAT under long-term fasting as a defensive reaction to inhibit the reduction of body temperature through an increase in PPAR-gamma activity.     

Physical separation of chlorophyll-protein complexes

This minireview focuses on the physical separation of chlorophyll-protein complexes and particles from the two photosystems by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) or differential/sucrose density-gradient centrifugation during the decade of 1960s. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suppression of Helicobacter pylori-induced gastritis by green tea extract in Mongolian gerbils

Since urease of Helicobacter pylori is essential for its colonization, we focused attention on foodstuffs which inhibit the activity of this enzyme. Among plant-derived 77 foodstuff samples tested, some tea and rosemary extracts were found to clearly inhibit H. pylori urease in vitro. In particular, green tea extract (GTE) showed the strongest inhibition of H. pylori urease, with an IC(50) value of 13 microg/ml. Active principles were identified to be catechins, the hydroxyl group of 5(')-position appearing important for urease inhibition. Furthermore, when H. pylori-inoculated Mongolian gerbils were given GTE in drinking water at the concentrations of 500, 1000, and 2000 ppm for 6 weeks, gastritis and the prevalence of H. pylori-infected animals were suppressed in a dose-dependent manner. Since the acquisition by H. pylori of resistance to antibiotics has become a serious problem, tea and tea catechins may be very safe resources to control H. pylori-associated gastroduodenal diseases.     

Purification,reconstitution, and characterization of Na(+)/serine symporter,SstT, of Escherichia coli

A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.     

Differences in MHC class I genes between strains of rainbow trout (Oncorhynchus mykiss)

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA, for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A-F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.     

Family 19 chitinase from Aeromonas sp. No.10S-24: role of chitin-binding domain in the enzymatic activity

Family 19 chitinase from Aeromonas sp. No.10S-24 (72.6 kDa) is composed of two chitin-binding domains (ChBDs), two proline- and threonine-rich (PT-rich) linkers, and a catalytic domain. The purified enzyme was labile in a standard buffer condition and spontaneously degraded into a 46-kDa fragment upon storage at 4 degrees C. The N-terminal sequence of the 46-kDa fragment was found to correspond to the sequence of the C-terminal region of the second PT-rich linker, indicating that the 46-kDa fragment is produced by truncation of the two ChBDs and the two PT-rich linkers from the mature protein, and consists only of the catalytic domain. The hydrolytic activities toward insoluble and soluble substrates were significantly reduced by the truncation of two ChBDs. In addition, antifungal activity determined from the digestion rate of haustoria of powdery mildew was reduced by the ChBD truncation. Although the profile of the time-course of N-acetylglucosamine hexasaccharide [(GlcNAc)6] degradation catalyzed by the ChBD-truncated enzyme was similar to that of the mature enzyme protein, the specific activity of the ChBD-truncated enzyme determined from the rate of hexasaccharide degradation was lower than that of the mature enzyme. The two CBDs appear to be responsible for facilitating the hydrolytic reaction. The sugar residue affinities (binding free energy changes) at the individual subsites, (-2) (-1) (+1) (+2) (+3) (+4), were estimated by modeling the hexasaccharide hydrolysis by the mature and ChBD-truncated enzymes. The truncation of ChBDs was found to strongly affect the affinity at the (-1) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.