Oligosaccharides involved in glycoconjugates play important roles in a number of biological events. To elucidate the biological functions of oligosaccharides, sufficient quantities of structurally defined oligosaccharides, are of limited availability by traditional purification methods, are required. Hence, chemical and enzymatic syntheses of oligosaccharides are becoming increasingly important in glycobiology and glycotechnology. In addition, oligosaccharides often occur as glycoconjugates attached to proteins or lipids. Hence, the development of simple and effective methods for synthesizing neoglycoconjugates such as neoglycoprotein and neoglycolipids is essential for an understanding of the biological function of these molecules. Here we review the most recent developments in the enzymatic synthesis of oligosaccharides and neoglycoconjugates.

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Since the first introduction of the sulfa drugs and penicillin into clinical use, large numbers of antibiotics have been developed and hence contributed to human health. But extensive use of antibiotics has raised a serious public health problem due to multiantibiotic resistant bacterial pathogens that inevitably develop resistance to every new drug launched in the clinic. Consequently, there is a pressing need to develop new antibiotics to keep pace with bacterial resistance. Recent advances in microbial genomics and X-ray crystallography provide opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics respectively. To prevent and control infectious diseases caused by multiantibiotic resistant bacteria, we need to understand more about the molecular aspects of the pathogens’ physiology and to pursue ways to prolong the life of precious antibiotics.

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Although dysfunctional telomeres and oncogenic or stressful stimuli are known to trigger cellular senescence in normal human diploid cells, the molecules and signaling network involved in the cellular senescence program are not fully understood. We have been trying to identify cellular senescence-inducing factors by various means. First, we screened for an extrinsic signal that can induce cellular senescence in human lung adenocarcinoma cell line A549, and identified transforming growth factor-β (TGF-β) as the cellular senescence-inducing factor. Cancer cells senesced by treatment with TGF-β impaired tumorigenicity both in vitro and in vivo, suggesting that cellular senescence functions as a tumor suppression mechanism. Next, we identified 86 independent senescence-associated genes by subtractive screening using A549-derived cell lines. Thirdly, we established novel cell lines (AST cells) from A549 cells exposed to mild oxidative stress. AST cells demonstrated functional impairment of telomerase due to perturbed subcellular localization of human telomerase reverse transcriptase, suggesting that mild oxidative stress might affect the cell fate of cancer cells. These results should provide insight into the molecular basis of the cellular senescence program.

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Breviones, allelopathic agents that have been isolated from Penicillium sp., are structurally unique diterpenoid derivatives. Breviones have attracted attention due to their bioactivity, because their allelopathic activities may offer agricultural use as environmentally benign herbicides. On the other hand, their structural uniqueness is also remarkable, and construction of their unique structure is a challenge from the viewpoint of organic synthesis. This review summarizes synthetic studies on breviones and structurally related natural products.

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The methylenedioxyphenyl moiety in the structure of sesamin and episesamin was changed into the catechol moieties, (1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, (1R,2R,5R,6S)-2-(3,4-dihydroxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, (1R,2R,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, (1R,2S,5R,6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, and (1R,2R,5R,6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane, in supercritical water. These products had same structures as the sesamin metabolites which act as antioxidants in the liver. These features suggested the direct preparation of antioxidants from sesamin by a one-step reaction using supercritical water.

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The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)₆-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)₆-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS–PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 °C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 °C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 °C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg²⁺, was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.

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Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K_eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K_eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.

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Clostridium paraputrificum M-21 β-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K_m values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.

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Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS–PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70–80 °C (k_cat⁄K_m=8.3 mM⁻¹ s⁻¹). In comparison with that of P. furiosus CP (k_cat⁄K_m=667 mM⁻¹ s⁻¹), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.

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The glucoamylase-encoding gene (glaB) promoter should be very useful for recombinant protein production in solid-state fermentation (SSF) of Aspergillus oryzae. A 97-bp fragment containing the cis-element of the glaB promoter was inserted into the glaA promoter, which was little expressed in SSF. The chimeric promoter showed about a 24-fold increase in promoter activity in SSF. Eight copies of the 97-bp fragment were tandemly fused with the glaB promoter. The improved promoter showed about a 4.6-fold increase in promoter activity in SSF. The glaB gene was overexpressed under control of the improved glaB promoter in SSF. Recombinant glucoamylase production reached about 1524 mg/kg-broth for 2 d. The improved glaB promoter should be very useful for overproduction of a recombinant protein in SSF of A. oryzae.

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A cDNA fragment encoding a 24 kDa allergenic protein in tartary buckwheat was obtained using reverse transcription PCR, 3′-rapid amplification of cDNA ends (RACE), and nest PCR. The cDNA clone contained 768 nucleotides, including 588 nucleotides in the open reading frame (ORF) and 180 nucleotides in the 3′-terminal sequence. The ORF encoded a functional protein of 195 amino acids. It shared 95% and 93% nucleotide homology with the allergenic storage protein and a legumin-like protein from common buckwheat respectively. The encoding region was expressed in host strain Escherichia coli BL21 (DE3) induced by IPTG at 28 °C. The inclusion bodies of recombinant protein obtained were analyzed by western blot and purified by affinity chromatography. The purity of target protein reached above 95%. After they were refolded by step-wise dialysis, 68% of the inclusion bodies reached soluble state. An analysis of immunological activity showed that the recombinant protein had a specific IgE binding activity. This is the first report of the molecular cloning and expression of the major allergen from tartary buckwheat.

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The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional α-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to α-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial α-amylases, but not toward animal or insect α-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.

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Four xylanases belonging to glycoside hydrolase family 10—Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulomonas fimi Cex (CF)—were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric β-1,4-linked xylopyranose as a precipitate during reaction with α-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X₂ as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.

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An essential ε-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 °C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.

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We investigated the change in renal mdr1b mRNA expression in offspring exposed to tributyltin chloride (TBTC) via the placenta and lactation or via lactation, using the real-time reverse transcription-polymerase chain reaction. Pregnant ICR mice were given water containing TBTC (0, 15, and 50 μg/ml) ad libitum from the start of pregnancy to weaning or from parturition to weaning. Exposure via the placenta and lactation significantly reduced the renal mdr1b level in offspring. Exposure to TBTC through the mother might impair the exclusion system of toxic compounds in offspring.

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A keratinolytic alkaline proteae (NAPase) from Nocardiopsis sp. TOA-1 degraded a scrapie prion without any chemical or physical treatment. Optimal temperature and pH were 60 °C and above pH 10.0. The scrapie prion was completely degraded within 3 min under optimal conditions.

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The aim of the current study was to investigate the metabolism of (−)-menthone by liver microsomes of humans. (−)-Menthone (1) was metabolized to (+)-neomenthol (2) (3-reduction) and 7-hydroxymenthone (3) by human liver microsomes. The metabolites formed were analyzed on GC and GC–MS. Kinetic analysis showed that K_m and V_max values for the metabolized (−)-menthone to respective (+)-neomenthol and 7-hydroxymenthone by liver microsomes of human sample HG70 were 0.37 mM and 4.91 nmol/min/mg protein and 0.07 mM and 0.71 nmol/min/mg protein.

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Fungal protease inhibitor F (FPI-F) from silkworm inhibits subtilisin and fungal proteases. FPI-F mutants P₁ residues of which, Thr²⁹, were replaced with Glu, Phe, Gly, Leu, Met, and Arg, were prepared. The inhibitory activities of mutated FPI-F against subtilisin and other mammalian proteases indicated that FPI-F might be a specific inhibitor toward subtilisin-type protease.

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O-Methyltransferase, POMT-9 was expressed in Escherichia coli. HPLC analysis of reaction products revealed three peaks corresponding to isoscopoletin, scopoletin, and scoparone, and their structures were determined using NMR. Biotransformation of esculetin with E. coli expressing POMT-9 generated scopoletin, isoscopoletin, and scoparone at 30.3, 21, and 31 μM respectively. POMT-9 is the first O-methyltransferase that produces three different O-methylated products.

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To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS–PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-β-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.

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Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 μM, 0.50 μM, and 0.48 μM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 μg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.

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We investigated the regulatory effects of structural differences among methylxanthine derivatives on the elevation of body fat percentage in developmental-stage rats. Caffeine, theophylline and theobromine were used as the methylxanthines. High-fat diets (20% lard) containing each methylxanthine (0.025%) were administered to male Sprague-Dawley rats for 12 weeks, with the result that the body fat percentage was generally reduced in each methylxanthine-fed group. The abdominal adipose tissue weight in the caffeine group was also significantly lower than that in the control group, the serum cholesterol and triglyceride levels in the caffeine group also being significantly lower than the levels in the control group. The study results suggest that caffeine could contribute most to preventing arteriosclerotic diseases.

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The accumulation of γ-aminobutyric acid (GABA) in the rice germ by using protease was investigated. After the proteolytic hydrolysis of germ protein by trypsin, the amount of GABA reached about 2.26 g per 100 g of germ. This demonstrates that the GABA yield could be significantly increased by protease. Our method is efficient and safe for manufacturing health food enriched with GABA.

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In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, μ-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of μ-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.

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To clarify the effects of storage temperature on potato components and acrylamide in chips, tubers from five cultivars were stored at various temperatures (2, 6, 8, 10, and 18 °C) for 18 weeks, and the contents of sugars, free amino acids in tubers, and acrylamide in chips after frying were analyzed. At temperatures lower than 8 °C, the contents of reducing sugars increased markedly in all cultivars, with similar increases in the acrylamide level and dark brown chip color. Free amino acids showed little change at the storage temperatures tested and varied within certain ranges characteristic of each cultivar. The contents of reducing sugars correlated well with the acrylamide level when the fructose/asparagine molar ratio in the tubers was <2. When the fructose/asparagine ratio was >2 by low-temperature storage, the asparagine content, rather than the reducing sugar content, was found to be the limiting factor for acrylamide formation.

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The purpose of this study was to examine the effects of dietary indigestible components on mucin secretion in the respective parts of the gastrointestinal tract through their physico-chemical properties. Rats were fed either a control diet or diets containing 5% polystyrene foam (PSF), 5% fructooligosaccharide (FOS), 5% PSF + 5% FOS, or 10% beet fiber for 10 d. Mucins in the small intestine and feces were greater in the PSF, PSF + FOS, and beet fiber groups than in the control and FOS groups. In the cecum, greater mucins were observed in the FOS, PSF + FOS, and beet fiber groups than in the control and PSF groups. None of the dietary treatment was effective on gastric mucins. Cecal mucins were significantly correlated with the cecal pool sizes of total short-chain fatty acids. The correlation between fecal mucins and fecal numbers was also significant. The results suggest that the effect of the bulk-forming property of the dietary indigestible component on mucin secretion is limited to the duct, while fermentability is effective only in the cecum.

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Many polysaccharides isolated from mushroom are considered to be biological response modifiers and have been shown to enhance various immune responses in vivo and in vitro. We demonstrate that a novel polysaccharide–protein complex (PPC) extracted from Phellinus linteus was a potent immunomodulator. PPC had a molecular weight of approximately 73 kDa. It was composed of five different monosaccharides, predominantly D-glucose and D-mannose, in the molar ratio of 3:2, the main amino acid being aspartic acid. PPC had a unique mode of immunostimulation with regard to its cell-type specificity. PPC was found to markedly increase the proliferation of B cells, but not T cells. Although PPC and lipopolysaccharide (LPS) had a similar mode of action in B cells, they were differentiated by the fact that PPC-induced cellular activation was not inhibited by polymyxin B (PB), a specific inhibitor of LPS. PPC increased the cytokine production and nitric oxide (NO) from macrophages. PPC also enhanced the lytic death of NO-sensitive tumor cells, B16 melanoma, through the production of NO. In addition, PPC up-regulated the natural killer (NK) cell-mediated killing of tumor cells, YAC-1 lymphoma in vitro. These results suggest that PPC stimulated the tumoricidal activities of macrophages and NK cells, and induced the proliferation of B cells in vitro. This process may be the mechanism by which PPC produced its therapeutic effects.

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The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were β-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit.

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Changes in the localization of calpain in conditioned and pressurized bovine skeletal muscles were investigated by immunogold electron-microscopy. In the muscle immediately after thawing (control), the relative distributions of colloidal particles statistically calculated by counting the colloidal particles were about 65% and 35% in the I-band/Z-disk and A-band regions, respectively. In the muscle conditioned for 7 days, distribution of colloidal particles was more than two times greater in both the I-band/Z-disk and A-band regions than in the control muscle. Almost no change in either the absolute concentration or relative distribution of the colloidal particles was detectable during further storage. In the muscle exposed to a pressure of 100 MPa or 200 MPa, slightly more immunogold was detected in the sarcomere than in that of the control muscle. Increasing pressure up to 300 MPa enabled high-density particles to be seen throughout the sarcomere. Conversely, few particles were detected in the sarcomere of the muscle exposed to 400 MPa. These electron-microscopic observations were confirmed from the statistical analysis as with the conditioned muscles.
It was clear from the results obtained that the pressure-induced changes in calpain localization were much more pronounced than those from conditioning.

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The antidiabetic effect of dahi was observed on high-fructose-induced diabetic rats. The fasting blood glucose, glycosylated haemoglobin, insulin, free fatty acids and triglyceride levels of the dahi fed group animals were significantly lower than those of the control group (p<0.05). The imparity of the glucose tolerance test was also delayed by one week in the dahi-fed animals.

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Leucinyl-arginyl-tryptophan (LRW) is a new peptide inhibitor of the angiotensin converting enzyme (ACE) that was previously predicted through quantitative structure-activity relationship modeling. LRW inhibited ACE activity in a competitive manner with a higher K_m value in the presence of the peptide, and the in vitro formation of angiotensin II by ACE was significantly reduced in the presence of LRW up to 60 min of incubation time.

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The hepatoprotective effects of whey protein on two injections of D-galactosamine (300 mg/kg, i.p.) were investigated in rats fed a modified AIN-93M diet formulated with a protein source of casein or whey for 16 d. The whey protein-containing diet clearly suppressed an increase in plasma alanine and aspartate aminotransferase activity, lactate dehydrogenase and bilirubin, which are hepatitis markers, and also hyaluronic acid, a fibrosis marker. In addition, it suppressed histopathological signs of portal fibrosis, bile duct proliferation, and perivenular sclerosis. These results suggest that supplementation with whey protein can help prevent the development of hepatitis and portal fibrosis.

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A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported.

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Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose–glycerol–peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.

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The sheath of Leptothrix cholodnii is a glycoconjugate composed of a polysaccharide and a peptide rich in cysteine. In this study, structural determination of the hydrazinolyzate of the sheath was carried out. Since the hydrazinolyzate is a polysaccharide incorporated with cysteine, it was S-derivatized with a thiol-specific fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Fluorescent fragments were purified by HPLC, and their structures were analyzed by mass spectrometry and NMR spectroscopy. The sheath was found to contain 2-(cysteinyl)amido-2-deoxy-D-galacturonic acid residue.

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