Inclusion of Amine Isomers with Open-Chain Hosts Having a Partial Structure of p-tert-Butylthiacalixarene.

Porous materials, which can capture a specific compound from a hard-to-separate molecular mixture, are strongly desired for practical separation and purification processes. Aiming to develop such materials, we have investigated the performance of our original host compounds, [3,3'-thiobis(5-tert-butyl-2-hydroxybenzene)-1,1'-diyl]diacetic acid (2) and its monopropyl ester (3), in discriminating among regio- or stereoisomers of three groups of amines, 2-, 3-, and 4-methylpyridine, 2-, 6-, and 8-methylquinoline, and cis- and trans-4-cyclohexanamine. Diacid 2 selectively included 4-methylpyridine in hexane and 3-methylpyridine in toluene in competitive inclusion among the three regioisomers. Mechanistic studies revealed that the inclusions of 3- and 4-methylpyridine are favored under kinetic and thermodynamic control, respectively. Solvent-dependent switching in guest selectivity was also observed in competitive inclusion among the methylquinoline isomers with diacid 2, whereas trans-4-methylcyclohexanamine was selectively included over the cis-isomer by monoester 3, as well as diacid 2, regardless of the solvent employed. X-ray crystallographic analysis of the resulting inclusion crystals suggests that the wide guest scope of the host compounds originates from their flexible ability to form complexes with amines.

Design and Synthesis of Open-Chain Hosts Having a Partial Structure of p-tert-Butylthiacalixarene.

The development of separation materials for hard-to-separate molecular mixtures is highly desired from environmental and economic perspectives. Although the crystal of p-tert-butylthiacalix[4]arene exhibits high guest selectivity in inclusion from a mixture of molecules with similar sizes and shapes, it cannot include molecules larger than its calix cavity. To extend its guest inclusivity, we designed and synthesized an open-chain host, [3,3'-thiobis(5-tert-butyl-2-hydroxybenzene)-1,1'-diyl]diacetic acid (4). The competitive inclusion among toluidine isomers using compound 4 gave inclusion crystals containing the p-isomer in 1:1 (host/guest) ratio, with lesser amounts of other isomers and/or solvent molecules. The isomer selectivity varied between 66% and 97% depending on the solvent employed. X-ray analysis of inclusion crystals 4·p-toluidine·MeCN and 4·p-toluidine·(o-toluidine)0.5 revealed that compound 4 includes p-toluidine by forming macrocyclic 2:2 inclusion complex(es) and that its higher-order structure has vacant spaces, in which molecules other than p-toluidine are included. Compound 4 was then transformed into monopropyl ester 5 to fill the vacant spaces with propyl moieties. Compound 5 included p-toluidine with high selectivity (∼96%) without the coinclusion of other molecules, regardless of the solvent employed.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.).

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed-protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano-LC-MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty-seven of the 152 proteins were associated with KEGG-defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed-protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma.

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preß1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preß1-HDL ELISA. Here, we purified preß1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preß1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preß1-HDL and original plasma preß1-HDL, or between the preß1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preß1-HDL MAbs, reactivity with intact plasma preß1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preß1-HDL is lipid-free monomolecular apoA-1.

Identification of molecular target of diallyl trisulfide in leukemic cells.

To identify the molecular target of diallyl trisulfide (DATS) in human leukemic cell line U937, we examined modification of thiol group(s) of cellular proteins by the redox 2D PAGE. A unique protein spot appeared by DATS treatment was identified to be heat shock protein 27 (HSP27). Hsp27 is suggested to be one of the molecular target of DATS in U937.

Identification of Nutritional Factors to Evaluate Periodontal Clinical Parameters in Patients with Systemic Diseases.

Nutritional factors reflect the periodontal parameters accompanying periodontal status. In this study, the associations between nutritional factors, blood biochemical items, and clinical parameters were examined in patients with systemic diseases. The study participants were 94 patients with heart disease, dyslipidemia, kidney disease, or diabetes mellitus. Weak negative correlation coefficients were found between nine clinical parameters and ten nutritional factors. Stage, grade, mean probing depth (PD), rate of PD 4−5 mm, rate of PD ≥ 6 mm, mean clinical attachment level (CAL), and the bleeding on probing (BOP) rate were weakly correlated with various nutritional factors. The clinical parameters with coefficients of determinations (R2) > 0.1 were grade, number of teeth, PD, rate of PD 4−5 mm, CAL, and BOP rate. PD was explained by yogurt and cabbage with statistically significant standardized partial regression coefficients (yogurt: −0.2143; cabbage and napa cabbage: −0.2724). The mean CAL was explained by pork, beef, mutton, and dark green vegetables with statistically significant standardized partial regression coefficients (−0.2237 for pork, beef, and mutton; −0.2667 for dark green vegetables). These results raise the possibility that the frequency of intake of various vegetables can be used to evaluate periodontal stabilization in patients with systemic diseases.

Effects of inorganic nitrogen sources on the production of PP-V [(10Z)-12-carboxyl-monascorubramine] and the Expression of the nitrate assimilation gene cluster by Penicillium sp. AZ.

A fungal strain, Penicillium sp. AZ, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12-carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that ammonia and nitrate nitrogen are available for PP-V biosynthesis, and that ammonia nitrogen was much more effective than nitrate nitrogen. Further, we isolated nitrate assimilation gene cluster, niaD, niiA, and crnA, and analyzed the expression of these genes. The expression levels of all these genes increased with sodium nitrate addition to the culture medium. The results obtained here strongly suggest that Penicillium sp. AZ produced PP-V using nitrate in the form of ammonium reduced from nitrate through a bioprocess assimilatory reaction.

Association between Dietary Habit and Clinical Parameters in Patients with Chronic Periodontitis Undergoing Supportive Periodontal Therapy.

The recurrence risk evaluation has been emphasized in periodontal stabilization during supportive periodontal therapy (SPT). However, nutritional factors, e.g., dietary habits such as the frequency of eating vegetables, are rarely included in the evaluation. In this study, the effect of nutritional factors on clinical periodontal parameters was examined in a lifestyle-related investigation and a periodontal examination in patients with periodontitis undergoing SPT. A total of 106 patients were recruited. Tendencies toward a negative correlation were found between rate of a probing depth (PD) of 4-5 mm, rate of PD ≥ 6 mm, the bleeding on probing (BOP) rate, periodontal inflamed surface area (PISA), and various nutritional factors. The number of teeth was a clinical parameter with a significantly high R2 (≥0.10) influenced by environmental factors, whereas PD, PD of 4-5 mm, the BOP rate, and PISA were influenced by nutritional factors. These results suggested that environmental factors reflected clinical parameters showing long-term pathophysiology, such as the PD rate. Nutritional factors tended to affect the current inflammatory pathophysiology, such as the BOP rate, PISA, and PISA/periodontal epithelial surface area. Therefore, environmental and nutritional factors appear to be useful for evaluating the risk of periodontitis during SPT.

Gut microbiota-dependent adaptor molecule recruits DNA methyltransferase to the TLR4 gene in colonic epithelial cells to suppress inflammatory reactions.

The intestine is inhabited by a large number of commensal bacteria that are immunologically non-self, potentially causing inflammation. However, in a healthy intestine, inflammation is strictly controlled at low levels to maintain homeostasis. We previously reported that the gut microbiota induce DNA methylation of the gene encoding Toll-like receptor (TLR) 4, a pattern recognition receptor that recognizes lipopolysaccharides of gram-negative bacteria, in colonic epithelial cells, suggesting its role in controlling intestinal inflammation. However, there remains a question of how gut microbiota cause methylation of only specific genes including TLR4, despite the fact that DNA methyltransferase (DNMT) is common to all genes targeted for methylation. Here, we identified RBM14 as an adaptor molecule that recruits DNMT to the TLR4 gene. RBM14 was shown to bind DNMT3 and be expressed at significantly higher levels in an intestinal epithelial cell (IEC) line with hypermethylated TLR4 gene than in an IEC line with hypomethylated TLR4 gene. In addition, RBM14 interacted with DNA regions of the TLR4 gene, and knockdown of RBM14 suppressed DNA methylation of the TLR4 gene in IECs. Furthermore, RBM14 expression was higher in colonic epithelial cells of conventional mice than in those of germ-free mice. Collectively, these results indicate that the gut microbiota induce methylation of the TLR4 gene in colonic epithelial cells by upregulating RBM14, which can recruit DNMT3 to the gene. The regulation of adaptor molecules such as RBM14, which bind to specific target genes and recruit DNMT, can explain, at least in part, how gut microbiota contribute to the maintenance of intestinal homeostasis through epigenetic control of specific gene expression in IECs.

Awamori fermentation test and 1-octen-3-ol productivity analysis using fatty acid oxygenase disruptants of Aspergillus luchuensis.

1-Octen-3-ol is a major aroma component of awamori, a traditional distilled liquor produced in Okinawa Prefecture, Japan. As 1-octen-3-ol is thought to affect the sensory properties of awamori, it is important to fully characterize the compound's biosynthetic pathway and control mechanism. We previously reported that the fatty acid oxygenase ppoC (ppo: psi-produced oxygenase) of Aspergillus luchuensis is directly involved in the production of 1-octen-3-ol in rice koji (Kataoka et al., J. Biosci. Bioeng., 129, 192-198, 2020). In the present study, we constructed A. luchuensis ppoD disruptants to characterize the role of ppo genes in 1-octen-3-ol biosynthesis. A small-scale awamori fermentation test was performed using ppoA, ppoC, and ppoD single disruptants (ΔppoA, ΔppoC, and ΔppoD, respectively), along with the parent strain, ΔligD. 1-Octen-3-ol was not detected in the distillate prepared using the ΔppoC strain. We conclude that A. luchuensis ppoC is the only 1-octen-3-ol-producing factor in the awamori brewing process. Because ΔppoA and ΔppoD slightly enhanced 1-octen-3-ol productivity, these two genes may play a role in negatively controlling 1-octen-3-ol biosynthesis.

Aspergillus luchuensis fatty acid oxygenase ppoC is necessary for 1-octen-3-ol biosynthesis in rice koji.

Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, 1-octen-3-ol was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.

Erythritol: Another C4 Platform Chemical in Biomass Refinery.

The potential of erythritol as a platform chemical in biomass refinery is discussed in terms of erythritol production and utilization. Regarding erythritol production, fermentation of sugar or starch has been already commercialized. The shift of the carbon source from glucose to inexpensive inedible waste glycerol is being investigated, which will decrease the price of erythritol. The carbon-based yield of erythritol from glycerol is comparable to or even higher than that from glucose. The metabolic pathway of erythritol biosynthesis has become clarified: erythrose-4-phosphate, which is one of the intermediates in the pentose phosphate pathway, is dephosphorylated and reduced to erythritol. The information about the metabolic pathway may give insights to improve the productivity by bleeding. Regarding erythritol utilization, chemical conversions of erythritol, especially deoxygenation, have been investigated in these days. Erythritol is easily dehydrated to 1,4-anhydroerythritol, which can be also used as the substrate for production of useful C4 chemicals. C-O hydrogenolysis and deoxydehydration using heterogeneous catalysts are effective reactions for erythritol/1,4-anhydroerythritol conversion.

Generation of Trichoderma reesei Mutant with Enhanced Xylanase Activity by Using Disparity Mutagenesis.

In the current study, we attempted to enhance the xylanase activity of Trichoderma reesei ATCC66589 by using disparity mutagenesis, wherein a plasmid harboring proofreading-impaired DNA polymerase δ was inserted. Following selection on xylan-rich media and successive plasmid curing, a mutant showing conidiospores strikingly different from those of the parent strain, with many small humped-surface spheres, was generated. Xylanase and ß-xylosidase activities of the mutant XM1, cultivated in xylan medium, were 15.8- and 11.0-fold higher than those of the parent strain, respectively. Furthermore, xylanase activity was generated approximately 24 h in advance compared to that in the parent. In contrast, when cultivated in Avicel medium, its xylanase and ß-xylosidase activities were 0.14- and 0.33-fold, respectively, compared to those in the parent. Among the xylan component sugars and related polyols, D-xylose and xylobiose exerted a distinct inductive effect on the xylanase activity in Avicel media, while xylitol and L-arabinose did not. Mutagenesis involved in xylose catabolism is suggestive of changes at the gene transcription level. Although the induction mechanism remains unclear in details, disparity mutagenesis may be useful for obtaining T. reesei mutants with high xylanase activity.

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4.

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].

Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4.

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using (1)H and (13)C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure: [structure: see text].

Complementary function of two transketolase isoforms from Moniliella megachiliensis in relation to stress response.

Two transketolase isogenes, MmTKL1 and MmTKL2, isolated from Moniliella megachiliensis were investigated for their roles in stress response and erythritol biosynthesis. The encoded proteins were highly homologous in amino acid sequence and domain structure. Two stress response elements (STREs) were found upstream of MmTKL1, while no STRE was found upstream of MmTKL2. In contrast, two Ap-1 elements were present upstream of MmTKL2, but none were detected upstream of MmTKL1. MmTKL2 partially complemented the aromatic amino acid auxotrophy of a Saccharomyces cerevisiae tkl1 deletion mutant, suggesting that at least one of the MmTKLs functioned as a transketolase in vivo. In response to short-term osmotic stress (20% glucose or 1.2 M NaCl) in Moniliella cells, MmTKL1 expression increased rapidly through the first 40 min before subsequently decreasing gradually, while MmTKL2 expression showed no significant change. In contrast, short-term oxidative stress (0.15 mM menadione) induced considerable increases in MmTKL2, while MmTKL1 expression remained low under the same conditions. Long-term osmotic stress (20% glucose) yielded increased expression of both genes starting at 12 h and continuing through 72 h. During either osmotic or oxidative stress, intracellular erythritol accumulation could clearly be correlated with the pattern of expression of either MmTKL1 or MmTKL2. These results strongly suggested that MmTKL1 is responsible primarily for the response to osmotic stress, while MmTKL2 is responsible primarily for the response to oxidative stress. Thus, we postulate that the two transketolase isoforms of M. megachiliensis play distinct and complementary roles in coordinating erythritol production in response to distinct environmental stresses.

Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.

Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.

PKCI-W forms a heterodimer with PKCI-Z and inhibits the biological activities of PKCI-Z in vitro, supporting the predicted role of PKCI-W in sex determination in birds.

The two chicken genes, PKCI-W on the W chromosome and PKCI-Z on the Z chromosome, belong to the gene family encoding the Hint (histidine triad nucleotide-binding protein)-branch proteins in the widely conserved HIT (histidine triad)-family. It has been speculated that PKCI-W is involved in the sex determination of birds by forming a heterodimer with PKCI-Z and inhibiting the function of PKCI-Z in female embryos. In this study, both PKCI-W and PKCI-Z were expressed in fusion [maltose-binding protein (MBP) or glutathione-S-transferase (GST)] and tagged [(His)(6) or FLAG] forms (FT-forms) in Escherichia coli and purified. Formation of homodimers of PKCI-W-containing or the PKCI-Z-containing FT-protein and the formation of a heterodimer between the PKCI-W-containing and the PKCI-Z-containing FT-proteins were demonstrated by Western blotting after GST-pulldown or binding to and elution from the Co(2+)-resin. The homodimer of PKCI-Z, but not PKCI-W, bound to an N(6)-(3- aminopropyl) adenosine affinity column and hydrolyzed adenosine 5'-monophosphoramidate. Both of these activities were inhibited in vitro in a dominant-negative manner by the formation of a heterodimer containing PKCI-W. These in vitro experimental results support the predicted role of PKCI-W in the process of sex determination in birds.

Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2.

A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).