The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.
To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS specta being measurable at as low as 200 pmol. Thus, this method consitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.
The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous “Annealed” polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated “Tendon” polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.
A low pH capillary electrophoresis (CE) was used for the measurement of free choline in plant leaves. Choline in the leaf extract was first converted to the benzoyl ester and put into CE. A well-resolved peak in the electropherogram was easily obtained. Involvement of enzymes in a two-step oxidation of choline to glycine betaine was evaluated in different plant species with the same mehod developed for glycine betaine and betaine aldehyde.
Hippospongic acid A (1) is a triterpene metabolite of the marine sponge, Hippospongia sp., with inhibitory activity against the gastrulation of starfish embryos. (R)-(+)-1 was synthesized by employing enzymatic kinetic resolution as the key step.
The structure of ostreocin D, a palytoxin analog isolated from the marine dinoflagellate Ostropsis siamensis, was found to be 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin by detailed 2D NMR analyses of intact ostreocin D and its ozonolysis products. Partial stereochemical assignments were done. This result indicates that the dinoflagellate O. siamensis is one of the biogenetic origins of palytoxin.
A polysaccharide with a molecular weight of 1.26×105, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1D, 2D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.
A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 μM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I-,Br-, and Cl- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.
The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified. It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa. To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn. Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT. Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p. Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p. The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin. The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.
Cry4A is a dipteran-specific δ-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.
Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.
Two lectins, Gymnothrax javanicus lectin-I (GJL-I) and Gymnothrax javanicus lectin-II (GJL-II) were isolated from the stomach and intestine, and the liver, respectively, of a toxic moray eel, Gymnothrax javanicus,. GJL-I is a polymer of two heterogeneous subunits of 67 and 51kDa. In a hemagglutination inhibition assay, it had sugar-binding spcificity toward lactose and lactulose among the mono- or oligo-saccharides and bovine submaxillary mucin (BSM) among the glycoproteins tested. The lectin stimulated nerve growth factor (NGF) synthesis by astroglial cells. GJL-II was a polymer of subunit of 41 kDa. This lectin had N-acetyllactosamine binding specificity.
We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans β-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo β-D-galactopyranoside (S6Galβ-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Galβ1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal β1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Galβ1-2Glc was mainly produced along with β-(1-1)α,β-(1-3),β-(1-6) isomers in 74% total yield. When methyl α-D-glucopyranoside (Glcα-OMe) was an acceptor, the enzyme also formed mainly S6Galβ1-2Glcα-OMe with its β-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of β-(1-2)-linked disaccharides. In contrast, with the corresponding methyl β-D-glucopyranoside (Glcβ-OMe) acceptor, S6Galβ1-3Glcβ-OMe and S6Galβ1-6Glcβ-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the β-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.
To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)nAla-OH (n=1 to 6), Fmoc-(Glu)nAla-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured. Km for Fmoc-peptides significantly decreased as peptide length increased from n=1 to n=5 with only slight changes in κcat. Km for Fmoc-(Glu)5,6Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull. Chem. Soc. Jpn., 73, 2587-2590). These results show that the enzyme has six subsites (S1' and S1-S5). Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted. For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate. It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1'-S1' interaction, so the amidase activity prevailed for Fmoc(Glu)3,5Ala-NH2. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.
Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular gycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35-39]. Extracellular glycoproteins have six distinct N-glycans (marked by *) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40% (Man5GlcNAc2, 28.8%;Man6GlcNAc2*, 6.4%; and Man7GlcNAc2*, 3.8%), while the plant-complex-type structures account for nearly 60% (GlcNAc2Man3Xyl1GlcNAc2*, 32.1%; GlcNAc1Man3Xyl1GlcNAc2 (containing two isomers)*, 6.2%; GlcNAc2Man3 GlcNAc2*, 4.9%; Man3Xyl1Fuc1GlcNAc2, 8.3%; and Man3Xyl1GlcNAc2, 3.7%).
In this study, we tried to determine(a) whether there is any permanent effect of oxLDL on the LCAT molecule and its activator lipoprotein apoA-I, and (b) the fate of oxLDL after its exposure to LCAT and apoA-I. Purified LCAT and LDL/oxLDL was incubated at 37°C for 1h, and separated by gel-permeation chromatography. Its activity was significantly less (30% less) after separating from oxLDL compared to that of LDL. Cofactor activity of isolated apoA-I (incubated with LDL/oxLDL) was found affected by oxLDL. These results indicate modification of the LCAT and apoA-I molecule. But LCAT was found more affected compared to apoA-I in terms of LCAT activity. We are also reporting a novel function of LCAT. It was found to reduce the adverse effects of oxLDL, for example, ability to affect the LCAT activity and TBARS value. This ability of LCAT to repair oxLDL was dose-dependent. But there was no change in its REM values or fluorescence intensity.
Hydroxyindole-O-methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.
In order to characterize EcoO109I methyltransferase, a recombinant Escherichia coli clone that overproduces the enzyme was constructed. The coding region of M.EcoO109I was joined to the lac promoter of an expression vector, pUC118, and the resulting plasmid was introduced into E. coli HB101. M.EcoO109I was purified homogeneously from IPTG-induced cells, and was found to consist of a monomer subunit. M.EcoO109I uniquely methylates the inner deoxycytidylate residue in the sequence 5'-(A/G)GGNCC(C/T)-3' to produce 5-methylcytosine. The enzyme was most active at pH 8.0-8.5 and 50°C. The enzyme activity was not affected by the addition of Mg2+ or EDTA.
A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expresion partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.
The effects of a large ascorbic acid dose on cytochrome P4501A1 gene expression induced by cigarette smoke exposure was studied in Osteogenic Disorder Shionogi rats, which lack ascorbic acid biosynthesis. The rats were divided into four groups and were administered either a minimal amount (4 mg/day, 4S and 4C) or a large amount (40 mg/day, 40S and 40C) of ascorbic acid. The 4S group and 40S group were daily exposed to cigarette smoke for 2 hours, while the 4C group and 40C group were not. At the end of the 25-day experiment, the rats were killed. The cytochrome P4501A1 mRNA level both in the liver and lung was measured by a competitive reverse transcription—polymerase chain reaction method. When a minimal amount of ascorbic acid was administered, the cytochrome P4501A1 mRNA increased in the liver of the cigarette smoke-exposed group (4S) compared with the control group (4C). On the other hand, when a large amount of ascorbic acid was administered, this increase was no observed in the cigarette smoke-induced group (40S) in liver. On the other hand, in lung, an increased mRNA level in 4S group was not decreased by large ascorbic acid administration (40S). This is the first direct mRNA-level evidence of the effects of a large ascorbic acid dose on the gene expression stimulated by cigarette smoke.
The effects of an extremely low frequency magnetic field (ELFMF) on the germination of plant seeds were examined. The decrease in the germination activity of the seeds of Arabidopsis thaliana WS kept in saturated humidity and high temperature (37°C) was suppressed by the exposure to a 400 mT ELFMF.
Allantoinase and allantoicase are known to form a complex in amphibian liver. In this study, a new type of allantoinase that did not form a complex with allantoicase was found in the amphibian liver. Purified enzyme had a molecular mass of about 44 kDa both in SDS-PAGE and gel-filtrations. The enzyme cross-reacted with anti-sardine allantoinase polyclonal antibody, and it weakly cross-reacted with anti-bullfrog allantoinase polyclonal antibody.
Changes in activity of RNA-degrading enzyme and amounts of phosphorus in mycelia and culture filtrate during Pi-sufficient and -deficient cultures of Pholiota nameko were investigated. The results showed that the intracellular and extracellular activities are controlled by the Pi concentration in the medium. Moreover, the induction and secretion of RNA-degrading enzymes under the Pi-deficient condition were analyzed by activity staining.
The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.
A previously uncharacterized yeast protein, YJL066c, was disovered in the membrane fraction although it has no hydrophobic stretch. The protein was partly solubilized by Triton X-100 in an oligomeric form, while it was insoluble in alkali or salt. By immunofluorescent microscopy, it localization coincided with the mitochondria. We therefore propose it should be named Mpm1 (mitochondrial peculiar membrane protein 1).
We investigated the content of four components of the O-2-producing enzyme (p47, p67, p22, and gp91) and the O-2-producing capacity in human myeloid cell lines. The content of the four components of the phagocyte oxidase was minimal before differentiation induction. During differentiation, expression of p22 and gp91 was at consistently low levels, even when the O-2-producing capacity was equivalent to that of normal neutrophils. On the other hand, p47 was consistently and rapidly induced to the level comparable to normal neutrophils. The results indicate that low expression of p22 and gp91 is sufficient to obtain normal O-2 production, and that p47 might play an important regulatory role in the functional differentiation.
The effects of Curcuma zedoaria, which is used as a condiment, in perfumery, and as a medicine, on immune response were investigated by measuring macrophage-stimulating activity in macrophages and RAW 264.7 cells. In this study, CZ-1 and CZ-1-III, the fractions partially purified from C. zedoaria, had a strong, dose-dependent lysosomal enzyme activity. It was suggested that active portions of CZ-1-III were polysaccharides rather than proteins. Phagocytic activity increased as a similar pattern in both the Gram-negative and Gram-positive bacteria, time-dependently. It was demonstrated that CZ-1-III can augment the oxygen burst response but had an even higher activity in vivo than in vitro. Also a significant increase of H2O2, NO, and TNF-α production was observed. However, the production of TNF-α at the concentration of 1,000 μg/ml decreased. These data suggested that C. zedoaria had macrophage-stimulating activity and the possibility of being used as a biological response modifier.
Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.
The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggegated. The release of soluble peptides from the aggregate was independent of the presence of chitosan. A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent. The results indicate that a milk agrregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.
The effects of capsaicin analogs on adrenaline secretion were investigated in rats. Capsaicin (20-100 μg/kg, iv) caused biphasic adrenalin secretion. Capsazepine 20 mg/kg, iv), a specific competitive antagonist of the vanilloid (capsaicin) receptor, strongly inhibited both phases of adrenaline secretion by capsaicin (50 μg/kg). Next, the effects of two capsaicin analogs on the adrenal catecholamine secretion were examined. Resiniferatoxin (20-200 ng/kg, iv), a naturally occurring phorbolester-like compound, provoked slow onset adrenaline secretion in a dose-dependent manner. Olvanil (2.46-246 μg/kg, iv), a synthesized non pungent capsaicin analog, also stimulated delayed catecholamine secretion dose-dependently. Capsazepine (20 mg/kg, iv) pretreatment prevented the resiniferatoxin (50 ng/kg)-and olvanil (24.6 μg/kg)- induced catecholamine secretion. These results suggest that some vanilloids (capsaicin, resiniferatoxin, olvanil) excite adrenaline secretion and such excitation is via the vanilloid receptor.
Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric α-amylase inhibitor as well as major allergens to baker’s asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying thepenetration characteristics of the CM proteins through intestinal mcosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministrered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.
Effects of bovine β-casein (1-28) having a phosphoserine-rich region (Glu14-SerP-Leu-SerP-SerP-SerP-Glu-Glu21) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1-28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG+IgM+IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated β-casein (14-21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine β-casein (1-28), but β-casein (14-21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides in attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.
To study whether the body fat-reducing potential of conjugated linoleic acid (CLA) could be increased through dietary manipulations, the effects of the combination of CLA with different proteins, fats, and sesamin were examined in rats. Male rats were fed diets containing 1% CLA or linoleic acid (LA) in combination with different proteins (20% of casein or soybean protein), fats (7% perilla oil or soybean oil) and 0.2% sesamin (SES) for 3 or 4 weeks. When the dietary fat source was soybean oil, CLA, as compared with LA, significantly reduced weights of epididymal and perirenal adipose tissues, irrespective of the dietary protein sources. However, the highest reducing effect was shown when soybean protein was given as a protein source. SES stimulated the reduction of epididymal and perirenal adipose tissue weights in both protein diets. In contrast, CLA increased the weight of brown adipose tissue, and SES further increased it in combination with soybean oil but not with perilla oil. No effect of dietary manipulation was observed on serum leptin and TNF-α levels. Thus, the body fat-reducing potential of CLA can be increased by an appropriate combination with food factors that may stimulate fatty acid β-oxidation.
Ajoene, a garlic-derived sulfur-containing compound, exhibited a hepatoprotective effect against acetaminophen-induced liver injury in mice. A pretreatment with ajoene suppressed the rise in serum glutamicpyruvic transaminase activity and the reduction in the hepatic reduced glutathione level. These effects of ajoene were observed dose-dependently (20-100 mg/kg). The pretreatment by ajoene also suppressed the decrease in hepatic protein thiol content resulting from acetaminophen administration.
Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-di-hydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.