There is a growing number of animal models and clinical trials of n-3 polyunsaturated fatty acid (PUFAs) supplementation in disease. Epidemiologic and biochemical studies have suggested beneficial effects of n-3 PUFAs. But also, the use of n-3 PUFAs has some potential toxicological risks that can be circumvented by careless processing, storing, and preserving the PUFAs. The use of n-3 PUFAs is safe if appropriate preparations and dosages are selected. Much research is needed to clarify their use under different disease conditions. The newly established clinical and nutritional facts on n-3 PUFAs will induce industry to develop food products based on this knowledge.
An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 188.8.131.52) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5•10-7-30•10-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.
Palatine chrome black 6BN (PCB6BN) is virtually non-fluorescent in an aqueous solution or in the presence of single-stranded DNA (ssDNA), whereas the fluorescence intensity of PCB6BN was linearly enhanced up to 300 μM of double-stranded DNA (dsDNA) base pairs. PCB6BN could be a useful fluorescent probe for quantifying dsDNA even when ssDNA is present for both heterogeneous and homogeneous assays.
Three yellowish compounds with 1,1-diphenyl-2-picrylhydrazyl-radical scavenging activity were newly isolated from the fermentation broth of Trichoderma sp. USF-2690 strain that had been isolated from a soil sample: two were novel oxidized sorbicillin dimers designated as bisorbibutenolide (1) and bisorbicillinolide (2), and one was sorbicillin (3) itself. The structures of 1 and 2 were determined from spectroscopic evidence. In the DPPH-radical scavenging assay, α-tocopherol gave an ED50 value of 17.0 μM after standing for 30 min, while continuing observation showed that the ED50 values for bisorbibutenolide, bisorbicillinolide, and sorbicillin slowly reached 80.8, 88.8 and 152.3 μM over 24 hr.
cis-p-Coumaroylagmatine (1) was isolated from Albizzia julibrissin Durazz, a nyctinastic plant, as a leaf-opening substance. The compound was quite effective for opening the plant leaves at 5×10-6 M at night, but was not effective for other nyctinastic plants. The bioactive fraction with leaf-closing activity was also separated from the plant extract. Although the leaf-opening activity of the plant extract changed between the day and night, the content of 1 was almost constant through a 24-h day. These results suggest that the change in content of an unknown leaf-closing factor induced balance between the two leaf-movement factors through a 24-h day.
4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone (EHMF) are known to inhibit cataract development in spontaneous cataract rats (ICR/f). Forty-five acylated hydroxyfuranone derivatives were designed and synthesized for an anti-cataract test, and their hydrophobic constants were also tested. Among these derivatives, 2,5-dimethyl-4-pivaloyloxy-3(2H)-furanone (HDMF pivalate) exerted a marked protective effect against the development of cataract in a galactose-induced model using cultured rat lens (in vitro). When tested on an ICR/f cataract model (in vivo), HDMF pivalate showed more significant inhibition of cataract development than parent compound HDMF. This derivative is more lipophilic than HDMF, so that HDMF pivalate can penetrate the cornea more easily than HDMF. The inhibition of cataract development by HDMF converted from HDMF pivalate is supported by the fact that HDMF was observed in the lens of ICR/f rats treated with HDMF pivalate.
Optically active α-methylbenzylphenylureas were synthesized and tested for their herbicidal activities against barnyardgrass and Cyperaceae paddy weeds in a greenhouse to evaluate the cross intergenus phytotoxicity between rice and barnyardgrass and the enantioselective phytotoxicity to the weeds. Several compounds controlled the growth of the weeds, and a suitable enantiomer for successful weed control was dependent on the type of weed and on the substituent at the aniline moiety. The (R)-2-isoPr and (R)-2-tert-Bu derivatives significantly controlled barnyardgrass and both annual and perennial Cyperaceae paddy weeds. The (R)-2-Et and (R)-2-CF3 derivatives showed the strong herbicidal activity against perennial Cyperaceae paddy weeds, while the (S)-enantiomers of the unsubstituted and fluoro derivatives were active against barnyardgrass. The enantioselectivity of the most potent compounds was high.
Macrophorins E, F and G were newly isolated from Botryosphaeria berengeriana, each showing potent antifungal activity similar to that of macrophorin A against phytopathogenic fungi B. berengeriana and Gibberella fujikuroi. Macrophorins E and G were identified as novel malonylated derivatives of macrophorin A, and F as a macrophorin E congener with a hydroxy-cyclohexenedione moiety instead of its epoxy-cyclohexenone one.
A newly rearranged 2(3→20)abeotaxane diterpenoid with a unique 6/10/6 skeleton was isolated from the bark of the Chinese yew, Taxus mairei. The structure was established as being 7β,13α-diacetoxy-2α,5α,10β-trihydroxy-9-keto-2(3→20)abeotaxane on the basis of 1-D and 2-D NMR data. The relative stereochemistry was defined from the results of a NOESY experiment. This is the first reported isolation of a rearranged 2(3→20)abeotaxane from Taxus mairei.
Two antimicrobial substances in rice hull were isolated and identified as 4-hydroxybenzoic acid and trans 4-hydroxycinnamic acid by LC-MS, and 1H- and 13C-NMR. An evaluation of 50% inhibition of growth (IC50) revealed that the two substances had different inhibition profiles against various microorganisms. Most of the gram-positive and some gram-negative bacteria were sensitive to trans 4-hydroxycinnamic acid and 4-hydroxybenzoic acid at IC50 concentrations of 100-170 and 160 μg/ml, respectively.
The nucleotide sequence of the largest double-stranded (ds) RNA (named dsRNA1) of three species of seed- and pollen-transmitted dsRNA species detected from Japanese pear was analyzed, and one strand was found to contain a single long open reading frame (ORF) of 1434 nucleotides that encoded a putative polypeptide containing 477 amino acid residues with a molecular mass of 54.9 kDa. This polypeptide contained amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Slow sedimentation of the dsRNA fraction suggests that the dsRNAs may be unencapsidated. The concentration of dsRNAs in the host, Japanese pear, was about 16 times higher than that from a cryptic virus, radish yellow edge virus (RYEV). These results suggest that the dsRNAs were not from cryptic viruses. Partial nucleotide sequences of the two smaller dsRNAs (named dsRNAs 2 and 3) and two other dsRNAs (named dsRNAs 4 and 5) detected from only the Japanese pear cultivar (cv.) Akita Tazawa 3 Gou were analyzed, and encoded nearly the same amino acid sequence encoded by dsRNA1.
A fragment of the starch-binding domain (SBDF) of Aspergillus niger glucoamylase was prepared using recombinant DNA techniques, and its thermal unfolding was investigated by high-sensitivity differential scanning calorimetry (DSC). Thermal unfolding of SBDF was found to be reversible at pH 7 as expected from a DSC study of the whole enzyme molecule [Tanaka A. et al., J. Biochem., 117, 1024-1028 (1995)] but not reversible at acidic region. Numerical analysis of the DSC curves showed that the denaturation was two-state, and some of the SBDF molecules were oligomeric (average degree of oligomerization was 1.2) at pH 7. It was suggested that the denaturation temperature of SBDF was lower than that of the starch-binding domain in the whole enzyme molecule by about 4.5 degree (decrease in the Gibbs energy change was 5.3 kJ mol-1) indicating a possibility that the starch-binding domain is stabilized by glycosylation of the domain itself, or by the highly glycosylated linker region.
Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methionine synthase cDNA was cloned and analyzed by RT-PCR, 3′- and 5′-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.
Aqualysin I is the alkaline serine protease isolated from an extreme thermophile, Thermus aquaticus YT-1. We analyzed kinetic properties of aqualysin I, using sixteen kinds of chromogenic succinyl-tripeptide p-nitroanilides as substrates. And we compared the substrate specificity of aqualysin I with those of proteinase K, subtilisin BPN′, and subtilisin Carlsberg. We found that aqualysin I had three subsites, S1, S2, and S3, in the substrate binding site. S1 site preferred alanine and phenylalanine. S2 site preferred alanine and norleucine. And S3 site preferred phenylalanine and isoleucine. These specificities were similar to those of proteinase K and subtilisin BPN′. The specificity of subtilisin Carlsberg differed from those of other enzymes.
N2733, 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride, was examined for its effect on TNF-α production by human myeloid THP-1 cells stimulated with lipopolysaccharide (LPS). N2733 inhibited LPS-induced release of TNF-α from THP-1 cells with an IC50 of 11 μM. N2733 did not affect the cell viability at the concentration of 50 μM or 100 μM. This indicates that N2733 is a potent inhibitor for TNF-α production without severe cytotoxicity. N2733 was also studied in two murine endotoxin shock models induced with LPS. One model was DBA/2 mice injected with LPS (5.6 mg/kg, i.v.), which increased the serum level of TNF-α within 1 hr. Treatment of these mice with N2733 (100 mg/kg×2, i.p.) decreased the serum level of TNF-α significantly. Another model was DBA/2 mice induced with LPS (30 mg/kg, i.v.), which reduced the survival rate to 30% during 7 days. Administrations of 30 mg/kg and 100 mg/kg N2733 (i.v.) restored the survival rates to 60% and 90% respectively. Our data demonstrate that N2733 inhibits LPS-induced TNF-α production, and this response is associated with an improvement in the survival rate of endotoxemic mice.
The precise substrate specificities of an α-L-arabinofuranosidase from Trichoderma reesei were investigated. The enzyme released arabinose at appreciable rates from p-nitrophenyl-α-L-arabinofuranoside, O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose (A1X2), arabinan, arabinoxylan, arabinogalactan, debranched-arabinan and gum arabic, but not from O-β-D-xylopyranosyl-(1→4)-[O-α-L-arabinofuranosyl-(1→ 3)]-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose (A1X3) or O-β-D-xylopyranosyl-(1→2)-O-α-L-arabinofuranosyl- (1→3)-O-β-D-xylopyranosyl-(1→4)-O-β-D-xylopyranosyl- (1→4)-D-xylopyranose (A1X4). The enzyme hydrolyzed methyl 2-O-, methyl 3-O- and methyl 5-O-α-L-arabinofuranosyl-α-L-arabinofuranosides to arabinose and methyl α-L-arabinofuranoside with the order of hydrolysis being: (1→5)->(1→2)-≥(1→3)-linkages. The enzyme hydrolyzed the (1→3)-linkage faster than the (1→5)- linkage of methyl 3,5-di-O-α-L-arabinofuranosyl-α-L-arabinofuranoside. The degree of conversion of arabinan and debranched-arabinan to monosaccharides by the enzyme was 33.0% and 9.1%, respectively. The α-L-arabinofuranosidase preferentially cleaved the arabinosyl sidechain from the arabinan rather than the terminal arabinosyl residue of the arabinan backbone.
A superoxide dismutase has been purified to apparent homogeneity from the muscular tissue of the ark shell, Scapharca broughtonii, by ammonium sulfate fractionation, and consecutive column chromatographies using DEAE-Sephadex and Sephadex G-100. This enzyme has a molecular weight of 71,700 and is composed of two identical subunits of Mr 35,800, which are joined by noncovalent interactions. The purified enzyme was stable over the range of pH 5.0-10.0 at 4°C for 24 h and at temperatures below 45°C. Cyanide at 0.1 and 1 mM inhibited the activity of the superoxide dismutase 56 and 100%, but 5 mM azide caused 8% inhibition. The optical spectrum of this enzyme had a maximum at 265 nm, and the amino acid composition of the enzyme was similar to that of the other Cu, Zn superoxide dismutases except for the contents of threonine, serine, proline, and leucine. Atomic absorption spectroscopy showed that this enzyme has approximately 2 atoms of Cu2+ and Zn2+ per mole of enzyme. These results indicate that the purified enzyme from ark shell, Scapharca broughtonii, is a Cu, Zn superoxide dismutase.
Pectin constituents, which were about 70 w/w% of extracellular polysaccharides (ECP) from a cell-suspension culture of Mentha, were purified by gel filtration chromatography, and their sugar composition and linkage were investigated. Two major constituents identified were (1→3)-linked galactan carrying arabinosyl residues on C-6 and (1→4)-α-linked galacturonan partially interspersed with (1→2)-linked rhamnosyl resides. Acetylated or methylated pectins were not identified on 1H-NMR analysis.
We investigated the conversion of D,L-2-amino-Δ2-thiazoline-4-carboxylic acid (D,L-ATC) to L-cysteine with Pseudomonas sp. ON-4a, an ATC-assimilating bacterium. Cysteine and N-carbamoylcysteine (NCC), but not S-carbamoylcysteine (SCC), were produced from D,L-ATC by a cell-free extract from the strain. These products were isolated from the reaction mixture and then identified as the L-form. Similar results were obtained with P. putida AJ3865 and unidentified strain TG-3, an ATC-assimilating bacteria. It became clear that L-NCC is an intermediate in the conversion of D,L-ATC to L-cysteine in these Pseudomonas strains. Furthermore, it was suggested that these bacteria have L-ATC hydrolase and L-NCC amidohydrolase.
Propolis was extracted using water and various concentrations of ethanol as solvents. The extracts were investigated by measurement of absorption spectrum with a UV spectrophotometer, reversed phase-high pressure thin-layer chromatography and reversed phase-HPLC. Maximum absorption of all extracts was 290 nm, resembling flavonoid compounds, and the 80% ethanolic extract showed highest absorption at 290 nm. The most isosakuranetin, quercetin, and kaempferol were extracted from mixtures of propolis and 60% ethanol, while 70% ethanol extracted the most pinocembrin and sakuranetin, but 80% ethanol extracted more kaempferide, acacetin, and isorhamnetin from propolis. The 60 to 80% ethanolic extracts of propolis strongly inhibited microbial growth and 70 and 80% ethanolic extracts had the greatest antioxidant activity and 80% ethanolic extract strongly inhibited hyaluronidase activity.
The Escherichia coli ArcB sensor is involved in anaerobic phosphotransfer signal transduction. ArcB is a hybrid sensor that contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. However, examination of the function of the newly-discovered HPt domain (named ArcBc) is still at a very early stage. To gain a general insight into the structure and function of the widespread HPt domains, on the basis of its three-dimensional crystal structure, in this study we constructed a certain set of mutants each having a single amino acid substitution in the HPt domain of ArcB. These ArcBc mutants were characterized and evaluated, based on the in vivo ability to signal the OmpR receiver via trans-phosphorylation.
Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.
Pomacea canaliculata α-mannosidase (260 kDa), composed of at least two isoforms with different pI, was partially purified. The activity was maximum at pH 4 and unaltered after incubation at 60°C for 60 min. ZnCl2, CaCl2, NaCl, and SH-reagents increased the activity, while MnCl2 and EDTA inhibited it. The enzyme catalyzed the hydrolysis of α1-2, α1-3, and α1-6 mannosidic linkages.
The phosphatase of a psychrophile (Shewanella sp.) was purified by ammonium sulfate fractionation, followed by sequential column chromatographies. The purified enzyme was electrophoretically homogeneous on native- and SDS-PAGE. Its molecular weight was 41,826 by its amino acid composition. The enzyme had its optimal pH for the activity at 9.8, and a broad substrate specificity to dephosphorylate ATP, pyrophosphate, glycerophosphate, and so on. Its activity was increased by metal ions including Mg2+, Mn2+, and Co2+. The maximal activity was observed at 40°C, and the enzyme at 0°C showed 39% of activity at 40°C. The enzyme, however, tended to lose its activity at 20°C and pH 9.8. These results indicated that purified enzyme was an alkaline phosphatase with characteristics; high catalytic efficiency at low temperature and gradual inactivation at an intermediate temperature.
An acid phosphatase with a very high substrate specificity for glucose-1-phosphate was isolated for the first time from mycelia of Pholiota nameko. The molecular weight of the enzyme was estimated to be 31,000 on gel filtration and 35,000 on SDS-PAGE. The activity was inhibited by Cu2+, Hg2+, molybdate, and tartaric acid. The sequence of N-terminal 20 amino acid residues was analyzed.
A mutant angiotensinogen, L11V, in which Val11 was substituted for Leu11 of ovine angiotensinogen was prepared to have the same scissile peptide bond as the human one. The mutation didn’t vary Km and kcat of human renin for the ovine substrate, but decreased those of rat renin to one half and one fortieth, respectively. Distances between the P1′ subsite of angiotensinogens and the 224th (human renin numbering) residue in the S1′ subsite of renins were estimated by molecular modelings. The marked decrease in kcat of rat renin for L11V could be attributed to the elongated distance between Val11 of L11V and Val221 of rat renin. It was also suggested that the distance is the reason why the human substrate cannot be cleaved by heterologous renins.
Complex formation of poorly water soluble organic compounds with cyclodextrin (CD) is quite difficult in an aqueous cyclodextrin system. Formation of the inclusion complex of d-limonene, phenyl ethanol, acetophenone, or menthol was investigated in a slurry form of α-, β-, or γ-CD in organic solvents or alcohol under anhydrous conditions. Ethanol and methanol were found to be good solvents for this method. The use of ethanol as the solvent was investigated in greater detail. There existed an optimal amount of ethanol for the maximum inclusion of d-limonene as the guest compound. However, an excess of ethanol inhibited the inclusion. An adsorption model of alcohol on CD, analogous to the substrate inhibition model of enzyme kinetics, could correlate the inclusion ratio with the amount of alcohol added to CD.
Bread dough was subjected to a high-voltage electric field (HVEF) during the first fermentation, and the bread firmness and the crystallinity of the starch (intensity of diffraction peak at 17.08° 2θ assigned to 4a; 5.24 Å d-spacing) isolated from the breads, which had been stored at 4 and 20°C, were examined. The HVEF treatment had the effects of reducing the bread firming at both storage temperatures as compared to the untreated bread. In this study, unexpected results were obtained for the crystallinity in the HVEF treated bread starches: while the firmness of the treated bread increased considerably after the first 3 days of storage at both temperatures, the rate of development in crystallinity was retarded at 20°C as compared with that of the untreated bread, but the opposite effect was observed at 4°C; that is, storing the bread at 4°C, the treated bread starch increased in crystallinity. These findings strongly suggest that crumb firming of the bread is involved in its water retention ability, taking into account the fact that the HVEF treatment made it possible to maintain bread softness longer than was possible for untreated bread. We, therefore, concluded that the increase in bread firmness was not closely related to the crystallinity of the bread starch, but was more influenced by the storage temperature.
The flavones apigenin and luteolin strongly inhibited the growth of HL60 cells and induced morphological differentiation into granulocytes. The flavonol quercetin inhibited the cell growth and induced a differentiation marker, i.e., NBT reducing ability. However quercetin-treated cells were not morphologically differentiated into granulocytes. The chalcone phloretin weakly induced NBT reducing ability and a marker of monocytic differentiation α-naphthyl butyrate esterase activity in the cells. Quercetin and phloretin appeared to induce the differentiation of HL60 cells into monocytes. The proportion of α-naphthyl butyrate esterase-positive cells induced by genistein was less than that of the NBT-positive cells. Some of the nuclei in genistein-treated HL60 cells morphologically changed. Genistein must have induced both granulocytic and monocytic differentiation of HL60 cells. The flavonols galangin and kaempferol, which had fewer hydroxyl group(s) in the B-ring than quercetin, and the flavanone naringenin inhibited the growth but did not induce the differentiation of HL60 cells.
Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.
A rapid stepwise measurement for the activities of calpastatin and μ- and m-calpains was developed by using 2-stage elution at pH 8.5 and then 7.0. The activities of calpastatin, μ-calpain and m-calpain can be rapidly assayed following the separation on DEAE-Sephacel chromatography by a 2 stage elution with 90 mM NaCl (pH 8.5), and then by 200 and 300 mM NaCl in elution buffer (pH 7.0). No significant differences in the recovery of these proteinases and inhibitor was observed between stepwise gradient and linear gradient methods.
We investigated effects of various tea infusions on mast cell activation using mouse mast cells. Among various tea extracts, infusions from cultivar ‘Benihomare’ and Taiwan lineage strongly inhibited histamine release after FcεRI cross-linking. Among three types of tea (from cultivar ‘Benihomare’), extract from oolong tea or black tea inhibited histamine release more strongly than green tea extract. Furthermore, ‘Benihomare’ oolong tea extract suppressed tyrosine phosphorylation of cellular proteins after FcεRI cross-linking, but polyvinyl polypyrrolidone treatment of the extract to remove phenolic compounds, weakened the suppressive effect.
The cytotoxicity of heterocyclic amines, dietary carcinogens derived from cooked foods, to primary cultured rat hepatocytes was studied. A tryptophan pyrolysis product, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was the most cytotoxic of 11 compounds tested. Trp-P-1 was found to induce apoptosis as measured by morphological changes in nuclear chromatin and internucleosomal DNA fragmentation. 3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) showed a moderate apoptotic effect, and other compounds had a similar but weaker effect.
Energy dispersive X-ray microanalysis in combination with scanning electron microscopy was used to examine the distribition of mineral nutrients in amaranth seed. It was found that P, K, and Mg were exclusively localized in embryonic tissue (cotyledons and radicles), but not in procambium. Since phytin globoids occur in cotyledons and radicles in the seed, it is conceived that these elements are associated with phytate. Sulfur was evenly distributed in the embryonic tissue including procambium, which might be derived from sulfur-containing proteins. Calcium was mostly present in seed coats and the boundary between the perisperm and embryo, suggesting that Ca is associated with pectins that constitute the network structure of cell wall.
A sodium chloride (NaCl)-sensitive mutant of Rhizobium fredii USDA191, which contained a single copy of Tn5-Mob transposed into chromosomal DNA, was obtained by Tn5-Mob random insertion. The growth rate of this mutant was lower than that of the wild type in the presence of 0.2 M NaCl and it seemed to lack the inductive ATP production in response to the addition of NaCl. This mutant induced the formation of small and whitish nodules on lateral roots of soybeans, which were negative for acetylene reduction activity, indicating that the nodules were ineffective for nitrogen fixation. The mutant also reduced the weight of above-ground portions and roots to 64 and 55%, respectively, compared with the weight of the plants inoculated with the wild-type cells. These results suggest that NaCl sensitivity of Rhizobium bacteria is one of the important factors for nodule formation and nitrogen fixation.
The psychrotrophic ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs). We found the function of a 26-kDa protein of the CSPs in the strain KUIN-1. In strain KUIN-1, a cold shock from 18 to 4°C induced the synthesis of the 26-kDa protein. By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4°C. The 26-kDa protein was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies (QA52, phenyl Superose, Superose 12, and Mono Q). The purified 26-kDa protein is composed of 6 subunits of 26.5-kDa with a molecular mass of approximately 159-kDa according to gel filtration and SDS-PAGE. The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-Gln-Gln-Val-Gln-Gln-His-Trp-Gly-His-His-. Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing. The characteristics of 26-kDa protein are analogous to the cold-regulated protein of the plants.
Chitosanases 33 kDa and 40 kDa in size were detected in the culture supernatant of Bacillus circulans WL-12. One of the two chitosanases, chitosanase 40 (40-kDa chitosanase) has been shown to be identical to the enzyme which has been reported previously as a β-1,3-1,4-glucanase by Bueno et al.1) The enzyme has been classified into family 8 glycosyl hydrolases together with the enzymes formally known as cellulase family D. This enzyme named chitosanase 40/β-1,3-1,4-glucanase hydrolyzed both chitosan and β-1,3-1,4-glucan with similar efficiency. However, the production of the enzyme was induced with chitosan but not by β-1,3-1,4-glucan. Therefore, it seems possible that the major substrate of this enzyme is chitosan rather than β-1,3-1,4-glucan. Analysis of degradation products generated from partially N-acetylated chitosan showed that chitosanase 40/β-1,3-1,4-glucanase hydrolyzes GlcN-GlcN and GlcN-GlcNAc linkages but not GlcNAc-GlcNAc nor GlcNAc-GlcN. The specificity for hydrolyzing linkages of this enzyme is similar to that of the chitosanase from S. griseus HUT6037.
In repetitive subcultures of Myxococcus xanthus cells, addition of penicillin to the developmental medium accelerated the appearance of the fruiting bodies and spore formation. During development of M. xanthus, DD-carboxypeptidase, which functions in the regulation of the level of cross-linkage, was also induced by penicillin. When D-alanyl-D-alanine, the structure of which is similar to the β-lactam structure of penicillin, was added to the developmental medium, 0.3 to 0.6 mM D-alanyl-D-alanine accelerated spore formation strongly. The activity of D-alanyl-D-alanine ligase, which synthesizes D-alanyl-D-alanine from D-alanine, increased gradually with maximum activity observed at the initial stage of sporulation. These results raise the hypothesis that M. xanthus produces and releases D-alanyl-D-alanine during development, and the released D-alanyl-D-alanine may function as an inducer of sporulation.
The structure of a sulfated polysaccharide-peptidoglycan complex (SP-PG) produced by Arthrobacter sp. was analyzed by NMR spectroscopy. In addition, oligosaccharide fragments of the SP-PG-L obtained by HF degradation were analyzed by NMR spectroscopy. These findings indicated that the sulfated polysaccharide (SP) contains a repeating unit composed of two galactofuranosides and a glucopyranoside. The main chain of the trisaccharide is [→6)β-D-Galf(1→6)-β-D-Galf(1→]n, with β-D-Glcp linked to one of the Galfs through a (1→2) linkage. The sulfated positions of the trisaccharide were identified as C-3 and C-5 of the β-glucosylated Galf residues, and C-2 or C-3 of the other Galf residue.
Three different carbohydrate-depleted enzymes were prepared from an endo-β-1,4-glucanase of Aspergillus niger IFO31125 by treatment with endo-β-N-acetylglucosaminidase or α-mannosidase. The molecular sizes of these enzymes decreased from 40 kDa containing about 8.9% carbohydrate to 39, 38, and 37 kDa with carbohydrate at 4.5, 1.3, and 0.8% (w/w), respectively. The surface net charges on these enzyme preparations were calculated from their electrophoretic mobilities measured by capillary zone electrophoresis. They had increased negative charges corresponding to the decreases in the carbohydrate content; those of native and 37-kDa enzymes were about -0.03 and -0.045, respectively. The surface hydrophobicities of proteins were also measured by partitioning the enzymes in a two-phase system containing polyethylene glycol and dextran, and decreased corresponding with decreases in their carbohydrate content. The results indicated that the high mannose type of carbohydrate chain in endo-β-1,4-glucanase affected the surface net charge on the enzyme and increased the surface hydrophobicity.
We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.
The gene encoding an extracellular lipase from Pseudomonas fluorescens No. 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.
Cellulose-producing Acetobacter xylinum has been known to secrete a cellulose-hydrolyzing β-1,4-endoglucanase (CMCax). When antibodies to recombinant CMCax were added to the culture medium, the formation of cellulose fiber was severely inhibited. Western blot analysis using the antibody showed that this enzyme was secreted into the medium even by a cellulose non-producing strain (Cel-). These results indicate that β-1,4-endoglucanase in the medium plays a critical role in the formation of cellulose fiber by the microorganism.
To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5′-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.