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The two main components from a Nelumbo nucifera leaf extract (NnEx) were investigated for their ability to prevent triglyceride accumulation and promoting lipolysis. Sun-dried Nelumbo nucifera leaves were immersed in hot water to extract the soluble components, and the resulting solution was analyzed by LC-MS and nuclear magnetic resonance. The results showed that quercetin-3-O-s-glucuronide (Q3GA) and quercetin were the key components of the NnEx. In vitro experiments confirmed that quercetin and Q3GA functioned in lipid metabolism by promoting triglyceride degradation through inhibition of the cAMP pathway. In vivo experiments showed that NnEx ingestion inhibited the accumulation of neutral fats in ICR mice and transitioned the hepatocytes of type II diabetic KK-Ay mice out of glycogenosis. These results highlight the ability of NnEx to control metabolism by modulating fat and sugar absorption and may provide an interesting novel treatment for obesity and related lifestyle diseases such as type II diabetes.

The two main components from a Nelumbo nucifera leaf extract (NnEx) were investigated for their ability to prevent triglyceride accumulation and promoting lipolysis. Sun-dried Nelumbo nucifera leaves were immersed in hot water to extract the soluble components, and the resulting solution was analyzed by LC-MS and nuclear magnetic resonance. The results showed that quercetin-3-O-ß-glucuronide (Q3GA) and quercetin were the key components of the NnEx. In vitro experiments confirmed that quercetin and Q3GA functioned in lipid metabolism by promoting triglyceride degradation through inhibition of the cAMP pathway. In vivo experiments showed that NnEx ingestion inhibited the accumulation of neutral fats in ICR mice and transitioned the hepatocytes of type II diabetic KK-Ay mice out of glycogenosis. These results highlight the ability of NnEx to control metabolism by modulating fat and sugar absorption and may provide an interesting novel treatment for obesity and related lifestyle diseases such as type II diabetes.

In this study, we performed microarray experiments using Lactobacillus delbrueckii subsp. bulgaricus 2038 cultured with or without Streptococcus thermophilus in whey medium to investigate nitrogen utilisation of these two strains, since Lb. bulgaricus 2038 use whey as their nitrogen before casein. For the utilisation of environmental free amino acids and peptides in co-culture, Lb. bulgaricus 2038 imported the types of peptides necessary for growth more effectively compared with monoculture because S. thermophilus primarily preferred different types of peptides than Lb. bulgaricus, but has lost the ability to prioritise the use of free branched-chain amino acids. Considering the ratio of nitrogen versus carbon being lower during co-culture, Lb. bulgaricus 2038 was induced to stop converting aspartate into carbon-skeleton intermediates which is catalysed by phosphoenolpyruvate carboxykinase. Despite mutualism, Lb. bulgaricus underwent competitive co-evolution with S. thermophilus to optimise nitrogen utilisation during co-cultivation.

Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lb. bulgaricus 2038) is an industrial bacterium that is used as a starter for dairy products. We proposed several hypotheses concerning its industrial features previously. Here, we utilized RNA-seq to explore the transcriptome of Lb. bulgaricus 2038 from four different growth phases under whey conditions. The most abundantly expressed genes in the four stages were mainly involved in translation (for the logarithmic stage), glycolysis (for control/lag stages), lactic acid production (all the four stages), and 10-formyl tetrahydrofolate production (for the stationary stage). The high expression of genes like d-lactate dehydrogenase was thought as a result of energy production, and consistent expression of EPS synthesis genes, the restriction-modification (RM) system and the CRISPR/Cas system were validated for explaining the advantage of this strain in yoghurt production. Several postulations, like NADPH production through GapN bypass, converting aspartate into carbon-skeleton intermediates, and formate production through degrading GTP, were proved not working under these culture conditions. The high expression of helicase genes and co-expressed amino acids/oligopeptides transporting proteins indicated that the helicase might mediate the strain obtaining nitrogen source from the environment. The transport system of Lb. bulgaricus 2038 was found to be regulated by antisense RNA, hinting the potential application of non-coding RNA in regulating lactic acid bacteria (LAB) gene expression. Our study has primarily uncovered Lb. bulgaricus 2038 transcriptome, which could gain a better understanding of the regulation system in Lb. bulgaricus and promote its industrial application.

A fluid-retention effect is required for beverages that are designed to prevent dehydration. That is, fluid absorbed from the intestines should not be excreted quickly; long-term retention is desirable. Here, we focused on the effect of milk protein on fluid retention, and propose a new effective oral rehydration method that can be used daily for preventing dehydration. We first evaluated the effects of different concentrations of milk protein on fluid retention by measuring the urinary volumes of rats fed fluid containing milk protein at concentrations of 1, 5, and 10%. We next compared the fluid-retention effect of milk protein-enriched drink (MPD) with those of distilled water (DW) and a sports drink (SD) by the same method. Third, to investigate the mechanism of fluid retention, we measured plasma insulin changes in rats after ingesting these three drinks. We found that the addition of milk protein at 5 or 10% reduced urinary volume in a dose-dependent manner. Ingestion of the MPD containing 4.6% milk protein resulted in lower urinary volumes than DW and SD. MPD also showed a higher water reabsorption rate in the kidneys and higher concentrations of plasma insulin than DW and SD. These results suggest that increasing milk protein concentration in a beverage enhances fluid retention, which may allow the possibility to develop rehydration beverages that are more effective than SDs. In addition, insulin-modifying renal water reabsorption may contribute to the fluid-retention effect of MPD.

The acquisition of amino acids by Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lb. bulgaricus 2038) when grown in the presence of bovine casein, the major protein in bovine milk, was investigated by examining the expression of genes related to proteolysis and amino acid biosynthesis. To support the growth on bovine casein, Lb. bulgaricus 2038 has to synthesise five kinds of amino acids de novo, as proteolysis from casein does not provide these. The incomplete hydrolysis in combination with amino acids biosynthesis may explain the slow growth of Lb. bulgaricus 2038 in a casein environment. Meanwhile, it was determined that Lb. bulgaricus 2038 uses different intracellular peptidases when grown in casein or whey medium, and initially yields the important amino acid glutamate from the C-terminal or N-terminal end of peptides imported into the cell.

We evaluated the effect of whey protein hydrolysates (WPH) on the water absorption rate in the small intestine using a rat small intestine perfusion model. The rate was significantly higher with 5 g/L WPH than with 5 g/L soy protein hydrolysates or physiological saline (p

Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038) is a bacterium that is used as a starter for dairy products by Meiji Co., Ltd of Japan. Culturing L. bulgaricus 2038 with whey as the sole nitrogen source results in a shorter lag phase than other milk proteins under the same conditions (carbon source, minerals, and vitamins). Microarray results of gene expression revealed characteristics of amino acid anabolism with whey as the nitrogen source and established a model of proteolysis and amino acid biosynthesis for L. bulgaricus. Whey peptides and free amino acids are readily metabolized, enabling rapid entry into the logarithmic growth phase. The oligopeptide transport system is the primary pathway for obtaining amino acids. Amino acid biosynthesis maintains the balance between amino acids required for cell growth and the amount obtained from environment. The interconversion of amino acids is also important for L. bulgaricus 2038 growth.

The amino acid biosynthesis pathway and proteolytic system of Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038), a mainstay of large-scale yogurt production, were modeled based on its genomic sequence. L. bulgaricus 2038 retains more potential for amino acid synthesis and a more powerful proteolytic system than other L. bulgaricus strains, but favors amino acid uptake over de novo synthesis. Free amino acids and peptides in bovine milk provide the main nitrogen sources; whey is more important than casein for L. bulgaricus during fermentation. Free amino acids are imported by amino acid permeases and by ABC-type transport systems whereas exogenous oligopeptides are imported by ABC-type proteins only. Histidine is neither synthesized nor imported singly, which might explain why L. bulgaricus cannot grow in synthetic media.

The amino acid biosynthesis and proteolytic system of Lactobacillus bulgaricus (L.Bulgaricus ) is important for its growth in niche-specific environments, as well as for flavour formation in the food industry. Comparative analyses of 4 completed sequences of the L.Bulgaricus strain genome on a genomic scale revealed that genes involved in amino acids synthesis were undergoing reductive evolution. However, the selected industrial strains, namely, L.Bulgaricus 2038 and L.Bulgaricus ND02, retained more complete genes in the amino acid synthesis and proteolytic system category than the laboratory strains, and have some unique genes and pathways for obtaining amino acids that enable these bacteria to adapt to their various environmental niches.

Phosphorylated dextran (P-Dex) is an acidic polysaccharide that functions as an immune adjuvant. P-Dex is known to regulate immune response by maintaining a balance between Th1 and Th2 cells in vitro, and thus may also be important in the control of allergic reactions. In the current study, we report the optimum conditions required for the efficient phosphorylation of dextran without toxicity. We found that when dextran was heated at 160°C for 24 h in phosphate buffer (pH 5.0), the resulting P-Dex demonstrated the highest phosphorus content (6.8%). We also report that P-Dex enhances mitogenic activity in mouse splenocytes and induces expression of CD69 and CD86 on the surface of B cells and dendritic cells (DC) in vitro. Oral administration of P-Dex to ovalubmin (OVA)-immunized mice was found to reduce antigen-induced cell proliferation and suppress the expression of CD86 on Th2-inducing DC via exogenous OVA stimulation. P-Dex was also found to increase IL-10 expression in the splenocytes of treated mice. These findings suggest that oral administration of P-Dex increases immunological tolerance and improves the specificity of immunological response to specific antigens.

Two angiotensin converting enzyme (ACE)-inhibitory peptides were isolated from enzyme modified cheese (EMC) and their amino acid sequences were identified as Leu-Gln-Pro and Met-Ala-Pro. The EMC was prepared by a combination of Protease N, Umamizyme, and Flavourzyme 500L. Both peptides were derived from β-casein, f 88-90 and f 102-104, respectively. Met-Ala-Pro showed strong ACE inhibitory activity (IC50=0·8 μm) and antihypertensive activity in spontaneously hypertensive rats (SHR) after single oral administration. The IC50value of Met-Ala-Pro was not affected by pre-incubation with ACE, suggesting that this peptide was a true ACE-inhibitory peptide. We report here, for the first time antihypertensive peptides from EMC.

Because the first step in the triggering of food allergy is the permeation of the allergen through the intestine, enhancement of the intestinal barrier function is thought to be effective for preventing food allergy. In this study, a peptide that inhibits ovalbumin (OVA) permeation in an in vitro Caco-2 cell model was isolated from enzymatic hydrolyzate of cheese (EHC). Amino acid sequence analysis identified the active peptide as GPIVLNPWDQ, a sequence identical to amino acids 102−111 of αs2-casein. The decapeptide significantly inhibited OVA permeation at a concentration of 10-6 M. In addition, it was found that a pentapeptide half, NPWDQ, is essential for the inhibitory activity because NPWDQ but not GPIVL had nearly the same inhibitory activity as GPIVLNPWDQ. The possibility exists that EHC and/or peptides possessing the NPWDQ sequence can be practically applied to the prevention of food allergy. Keywords: Enzymatic hydrolyzate of cheese; allergen; permeation; Caco-2; αs2-casein; ovalbumin

Background: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy. Methods: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2). Results: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG. Conclusion: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Bioguided fractionation of the methanol extract of Momordica charantia dried gourds led to the isolation of three new cucurbitane triterpenoids (1-3), together with eight known compounds (4-11). The aglycone of momordicoside I was isolated from the ether soluble fraction in a high amount. The structures of the metabolites were established on the basis of one and two dimensional NMR spectroscopic evidence, X-ray analysis, and comparison with the reported data in the literature. A number of phytochemicals have been isolated from Momordica charantia but the constituents responsible for the hypoglycaemic/antihyperglycaemic activities have not been determined. Therefore, in order to evaluate the contribution of the cucurbitane triterpenoids of the ether fraction of M. charantia methanol extract to in vivo anti-diabetic effects, the major compounds, 5beta,19-epoxy-3beta,25-dihydroxycucurbita-6,23(E)-diene (4), and 3beta,7beta,25-trihydroxycucurbita-5,23(E)-dien-19-al (5) have been tested and have shown blood hypoglycaemic effects in the diabetes-induced male ddY mice strain at 400 mg/kg. The two aglycones of charantin did not show any hypoglycaemic effects. Our finding is the first demonstration that major pure cucurbutanoid compounds of M. charantia have in vivo hypoglycaemic effects.

The murine macrophage line J774.1 was cultured in the presence of neutral polysaccharide (NPS) or phosphopolysaccharide (APS) produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1. Alteration of macrophage functions induced by the extracellular polysaccharides was evaluated by observing changes in the morphology of cells, nitrite production by cells, and gene expression of cytokines. Treatment with APS caused the cells to morphologically change into an active form, while there was almost no stimulation of the cells by NPS. Neither NPS nor APS could induce nitric oxide production, but lipopolysaccharide (LPS) could. The mRNA expression of several cytokines was greater in cells stimulated with APS than in cells stimulated with NPS. When the cells were stimulated with APS at a concentration of 200 μg/ml for 12 h, strong IL-1 α gene expression was detected. The expression of IL-6, IL-10, IL-12p40 and TNF- α on murine macrophages was also increased by APS, but that of IL-12p35 was decreased. It is concluded that APS produced by strain OLL1073R-1 enhances the functions of macrophages in a different manner from LPS.

Aqueous vanadate and aqueous tungstate have been known to mimic all or most of the actions of insulin in intact cell systems with respect to normalization of the blood glucose level. By carrying out oral administration in vivo experiments on the blood glucose level of streptozotocin (STZ)-induced diabetes (STZ mice), the insulin-mimetic (IM) effects of metal-oxide clusters of all-inorganic composition were examined using many types of polyoxometalates (POM) with and without vanadium substitution. Several homo-POM and vanadium-substituted POM showed hypoglycemic effects. The observed hypoglycemic effects indicated that POM with the Dawson structure [[alpha-P(2)W(18)O(62)](6-) (W-2), [alpha-P(2)W(17)V(V)O(62)](7-) (V-19) and [alpha-1,2,3-P(2)W(15)V(V)(3)O(62)](9-) (V-04)] are more effective than those with the Keggin structure [[alpha-PW(12)O(40)](3-) (W-1), [alpha-PW(11)V(V)O(40)](4-) (V-01), [alpha-1,2-PW(10)V(V)(2)O(40)](5-) (V-02), [alpha-1,2,3-PW(9)V(V)(3)O(40)](6-) (V-03) and [alpha-1,4,9-PW(9)V(V)(3)O(40)](6-) (V-13)]. The vanadate cluster [V(10)O(28)](6-) (V-15) also showed a hypoglycemic effect. (31)P and (51)V NMR measurements showed that the Dawson POM (W-2, V-04 and V-19) are stable in aqueous solution under the conditions used. The effect of all POM on the body weight of STZ mice was also examined. The decrease in body weight after administration of W-2 was much less than for V-19, V-04 and V-15. This suggests that not only monomeric tungstate and vanadate, but also the structure factors of tungstate and vanadate clusters, can play a significant role in their biological action.

The level of expression of midkine (MK), a heparin-binding growth factor, is increased in many types of human carcinomas. An enzyme-linked immunoassay, which utilizes a combination of rabbit and chicken antibodies revealed that serum MK level in the controls (n= 135) was 0.154 ± 0.076 (mean ± SD) ng ml–1with an apparent cut-off value as 0.5 ng ml–1. Serum MK level was significantly elevated in the cancer patients (n= 150) (P< 0.001); 87% of the patients showed levels of more than 0.5 ng ml–1. All ten types of cancer examined showed a similar profile of serum MK level. There was no or weak correlation between C-reactive protein level, a marker of inflammation, and serum MK level. Furthermore, in case of gastric carcinoma and lung carcinoma, patients with stage I carcinoma already showed elevated serum MK levels. The present results indicated that serum MK could serve as a general tumour marker with a good potential for clinical application. © 2000 Cancer Research Campaign

Two water-soluble, silver(I) complexes showing a wide spectrum of effective antibacterial and antifungal activities, i.e., {[Ag(Hhis)]·0.2EtOH}2 (1; H2his = l-histidine) and [Ag(Hpyrrld)]2 (3; H2pyrrld = (S)-(−)-2-pyrrolidone-5-carboxylic acid) were prepared. In aqueous solution 1 and 3 were present as dimers, whereas in the solid state they were polymers. Crystallization of 1 by slow evaporation and/or vapor diffusion gave water-insoluble crystals of [Ag(Hhis)]n (2) showing modest antimicrobial activities. The complex 1 in the solid state is a polymer formed by intermolecular hydrogen-bonding interactions between dimeric [Ag(Hhis)]2 cores, while 2 is a different polymer without a core complex. X-ray crystallography revealed that 2 was a left-handed helical polymer consisting of a bent, 2-coordinate silver(I) atom bonding to the Namino atom of one Hhis- ligand and the Nπ atom of a different Hhis- ligand. Of particular note is the fact that Ocarboxyl atoms do not participate in the coordination. X-ray crys...

Two types of silver(I)-triphenylphosphine complexes with 2-mercaptonicotinic acid (H2mna) were prepared as [Ag(Hmna)(PPh3)n] (1, n = 2; 2, n = 3); their crystal structures determined by single-crystal X-ray diffraction. These complexes were obtained from reactions of the precursor, [Ag(Hmna)]n, with less excess (3 equiv) and large excess (20 equiv) amounts of PPh3, respectively. Complex 1 was also obtained from a reaction among another precursor [AgCl(PPh3)3], H2mna and aqueous NaOH. The coordination geometry of 1 was a trigonal pyramid with an AgSP2 core; there was a weak interaction between the silver(I) center and the pyridine nitrogen atom, while that of 2 was a distorted tetrahedron with an AgSP3 core. In both 1 and 2, the carboxyl group was protonated and its oxygen atoms did not participate in the coordination. In contrast to the formation of 1 and 2, a related complex, [Ag(Hmba)(PPh3)3] 4 (H2mba = 2–mercaptobenzoic acid), has been obtained from a reaction of [Ag(Hmba)]n with a stoichiometric amoun...

Two novel coinage metal(I)-triphenylphosphine complexes with a heterocyclic ligand, [Ag(tetz)(PPh 3 ) 2 ] n ( 1 ) and [Au(tetz)(PPh 3 )] ( 2 ) (Htetz=tetrazole) were synthesized from a reaction in dichloromethane of the precursor complex [Ag(tetz)] n with PPh 3 and from a reaction in acetone of [AuCl(PPh 3 )] with Htetz in the presence of aqueous NaOH, respectively, and isolated as colorless needle crystals for both 1 and 2 . The crystal structures of 1 and 2 were determined by single-crystal X-ray diffraction. These d 10 complexes were also fully characterized by elemental analyses, TG/DTA and FT-IR in the solid-state, solution NMR ( 31 P, 1 H and 13 C, as well as 109 Ag for 1 ) spectroscopies and solution molecular weight measurements. Complex 1 , is composed of a helical polymer, a 2 1 helix with a pitch 9.471 A, formed by a bridged tetrazolate of an AgNP 2 core in the solid-state, but it was present as a monomer in solution. In contrast to 1 , complex 2 consisted of a monomeric two-coordinate AuNP core without gold(I)–gold(I) interaction both in the solid-state and in solution. The molecular structures of 1 and 2 were compared with those of the corresponding triazole complexes, i.e. silver(I) complexes [Ag(1,2,3-triz)(PPh 3 ) 2 ] n ( 3 ) and [Ag(1,2,4-triz)(PPh 3 ) 2 ] n ( 4 ) both as helical polymers, a monomeric gold(I) complex [Au(1,2,3-triz)(PPh 3 )] ( 5) and a dimeric gold(I) complex [Au(1,2,4-triz)(PPh 3 )] 2 ( 6) through an intramolecular gold(I)–gold(I) bond; the structures of complexes 3 – 6 being recently determined by X-ray crystallography. The antimicrobial activities of 1 , its precursor [Ag(tetz)] n and 2 were compared and key factors affecting them discussed.

A neutral, Ag(I)N bonding compound, polymeric silver(I)-imidazolate [Ag(imd)] n (1) consisting of Ag + : imd = 1:1 (Himd = imidazole, C 3 H 4 N 2 ), whowed wide spectra in effective antimicrobial activities against bacteria, yeast and mold. Of particular note are the activities against a wide range of mold. This polymeric solid does not crystallize and is sparingly soluble in all solvents. The monomeric, cationic, water-soluble Ag(I)N bonding complex, [Ag(Himd) 2 ](NO 3 ) (2), has also shown wide spectra of effective antimicrobial activities. These activities observed here were significantly different from those of the recently prepared oligomeric (Ag(I)S bonding complexes; the latter have shown narrow spectra. It is proposed that the Ag(I)N bonding is one of the key factors showing the wide spectra of antimicrobial activities and the potential targets for inhibition of bacteria and yeast by these Ag(I) complexes are proteins, but not nucleic acids. The physico-chemical properties of (1), in comparison with those of (2), with various measurements (FT-IR, Laser Raman scattering spectroscopy, ESCA and solid 13 C CP-MAS NMR spectroscopies) are described.

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine β-casein phosphopeptides (β-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-β-CPP(f(1–25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from β-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in β-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Sodium salt of anionic Au(I) complex with a trianion of thiomalic acid tma3− (H3tma = HOOCCH(SH)CH2COOH), known as an effective antiarthritic drug, was prepared in highly pure form by one-step synt...

Thiosalicylic acid ligand (H 2 TSA = o-HS(C 6 H 4 )COOH) forms two types of anionic light- and thermally stable Ag(I) complexes with remarkable antimicrobial activities for bacteria, yeast, and mold; one is a yellow, water-soluble complex 1 and the other an orange, water-insoluble complex 2 , either of which can be converted to the other, and both of which do not crystallize. Both are complexes with composition of metal: TSA 2− ligand = 1:1. The complex 2 is probably polymeric, whereas the complex 1 is an oligomer with a formula of {Na[Ag(TSA 2− )·H 2 O]} n (n = 12 − 14; MW = 3600 − 4200). Both complexes have been characterized with complete elemental analyses, TG/DTA, FT-IR, and ESCA spectra in the solid state, and complex 1 as well as with molecular weight measurement in aqueous solution, and with 109 Ag and 2D NMR( 1 H- 1 H COSY, 1 H- 13 C COSY, and HMBC) measurements in aqueous solution.

A water-soluble, thermally and light-stable, anionic AgI complex with a trianion of thiomalic acid, TMA3− (H3TMA = HOOCCH(SH)CH2COOH) as a ligand was prepared and isolated as a typically yellow-coloured powder of the sodium salt, which showed excellent antimicrobial activity against some bacteria, yeast and moulds. All attempts at crystallization were unsuccessful. This compound was characterized by complete elemental analyses, TG/DTA, FT-IR, various NMR (1H, 13C and 109Ag) spectroscopies, and molecular weight determination in aqueous solution by a combination of molmass measurement based on the cryoscopic method with degree of dissociation determination based on the [Na+] measurement in the equilibrium state using an Na+ ion-selective electrode. It was shown that this complex was not a monomeric species, but an oligomer with a formula of [NaH [Ag(TMA)]·0.5H2O]n(n = 15–19; MW = 4320–5470). Analogues of this anionic AgI complex were also obtained as potassium, barium, ammonium, lithium and caesium salts, as well as a free-acid form.

Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) is an important species of Lactic Acid Bacteria (LAB) used for cheese and yogurt fermentation. The genome of Lb. bulgaricus 2038, an industrial strain mainly used for yogurt production, was completely sequenced and compared against the other two ATCC collection strains of the same subspecies. Specific physiological properties of strain 2038, such as lysine biosynthesis, formate production, aspartate-related carbon-skeleton intermediate metabolism, unique EPS synthesis and efficient DNA restriction/modification systems, are all different from those of the collection strains that might benefit the industrial production of yogurt. Other common features shared by Lb. bulgaricus strains, such as efficient protocooperation with Streptococcus thermophilus and lactate production as well as well-equipped stress tolerance mechanisms may account for it being selected originally for yogurt fermentation industry. Multiple lines of evidence suggested that Lb. bulgaricus 2038 was genetically closer to the common ancestor of the subspecies than the other two sequenced collection strains, probably due to a strict industrial maintenance process for strain 2038 that might have halted its genome decay and sustained a gene network suitable for large scale yogurt production.

We have found that an enzyme-modified cheese (EMC) inhibited the permeation of allergens such as ovalbumin (OVA), using Caco-2 cells as an in vitro intestinal epithelial model. In addition, NPWDQ (Asn-Pro-Trp-Asp-Gln, aa 107-111 of alphas(2)-casein) was isolated from EMC and identified as one of the responsible peptides for this inhibitory activity (Tanabe et al., J. Agric. Food Chem., (2007)). In this study, we aimed to clarify the mechanism by which NPWDQ inhibited allergen permeation in vitro, and also to evaluate the effects of EMC on allergen permeation in vivo. Intestinal permeability for both fluorescein isothiocyanate conjugated dextran and horseradish peroxidase was decreased in Caco-2 cells by the addition of NPWDQ, indicating that NPWDQ might inhibit both paracellular and transcellular transports. Next, intestinal inflammation was induced by subcutaneous injections of indomethacin to rats. When OVA was injected into the jejunal and ileac loops of indomethacin-administered rats with and without NPWDQ, it was found that the addition of NPWDQ effectively diminished OVA permeation from both loops. Although the plasma OVA concentration of indomethacin-administered rats after oral OVA challenge was markedly elevated over that of normal rats, supplemental administration of EMC to the rats effectively suppressed OVA permeation. These results suggest that EMC is useful for the prevention of food allergy by inhibiting allergen permeation probably by enforcing the intestinal barrier.

Twenty lactobacilli isolated from human feces were studied for binding to the human blood type B-antigen [Galalpha1-3 (Fucalpha1-2) Gal-] and H-antigen (Fucalpha1-2Gal-] expressed sugar chains in human intestinal mucosa. We found two strains, L. gasseri OLL2755 and L. gasseri OLL2877 that firmly adhere to human B-antigen, and we found L. gasseri OLL2827 bound to the H-antigen.

Adherent lactic acid bacteria (LAB) in the human intestine were investigated using the surface plasmon resonance technique with the biosensor BIACORE-1000. Ninety-three LAB strains were isolated from human feces and evaluated for binding to human blood type-A antigen [GalNAcα1-3 (Fucα1-2) Gal-: A-trisaccharide] expressed in the intestinal mucosa. Eleven strains showed strong adherence to an A-trisaccharide biotinyl polymer (BP) probe, and slightly or no adherence to a B-trisaccharide BP probe. Four strains with high adherence (high A/B ratio) were selected and their surface layer proteins (SLPs) were evaluated for A-antigen ligand binding using BIACORE. The SLP from L. brevis strain OLL2772 showed a single band at ca. 48 kDa by SDS–PAGE analysis and it had a very strong adherence to the human A-antigen, as shown using an anti-A lectin blocking technique. A partial N-terminal sequence of the band showed strong homology to an S-layer protein of L. brevis ATCC8287T. The probiotic LAB binds to human blood type-A antigen expressed in the intestinal mucosa which may aid in colonization of the gut.

Non-Helicobacter bacteria can be cultured from the gastric mucosa in adults but in children, there are no studies about such microflora. The purpose of this study, therefore, was to clarify whether gastric biota develops in children. In 10 children and 10 adults or elderly (5 H. pylori-infected and 5 uninfected in each group), biopsy specimens of the gastric antrum and corpus and gastric juice were studied for bacterial examinations and the data were compared between both age groups in relation to H. pylori status and luminal pH. Bacterial genera and species were analyzed using both culture and real-time polymerase chain reaction (PCR) with the 52 genus- and species-specific primer sets. Non-Helicobacterbacteria in the mucosa were cultured from all adult patients, whereas microorganisms were cultured in only one child (p < .001). Gastric pH was lower in children (median, 1.4) than in adults (median, 2.6) (p < .005). The grade of endoscopic gastric atrophy was moderate or severe in 8 adults, but absent or mild in all 10 children. Among adults, there was a significant positive correlation between gastric pH and total bacterial counts of both the mucosa and juice. These data indicate that impaired gastric acid secretion associated with long-term H. pylori infection enables non-Helicobacter bacteria to colonize in the human stomach. Such microorganisms rarely colonize in the gastric mucosa in children regardless of H. pylori status.

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P

Midkine (MK) is a heparin-binding growth factor, which promotes growth, migration, and survival of various cells, and MK expression is increased in many human carcinomas. We determined the urinary MK level by enzyme-linked immunoassay. Taking 311pg/mg creatinine as a cut-off level, 70% of patients with various carcinomas (n=142) gave positive values, while only 5.5% of healthy volunteers (n=330) did. In case of gastric carcinoma, 17 out of 21 patients with stage 1 tumor were positive. Urinary MK levels are expected to become a convenient marker as an aid in detection of tumors.

Two isomeric gold(I)-triphenylphosphine complexes with nitrogen-containing heterocycles, [Au(L)(PPh3) (HL = pyrazole (1), imidazole (2)) were isolated as colorless cubic crystals for 1 and colorless plate crystals for 2, respectively. The crystal structures of 1 and 2 were determined by single-crystal X-ray diffraction. These complexes were also fully characterized by complete elemental analyses, thermogravimetric/differential thermal analyses (TG/DTA) and FT-IR in the solid state and by solution NMR (31P, 1H and 13C) spectroscopy and molecular weight measurements in acetone solution. These complexes consisted of a monomeric 2-coordinate AuNP core both in the solid state and in solution. The molecular structures of 1 and 2 were compared with those of related gold(I) complexes, [Au(1,2,3-triz)(PPh3)] (3, Htriz = triazole), [Au(1,2,4-triz)(PPh3)]2 (4) as a dimer through a gold(I)-gold(I) bond in the solid state, and [Au(tetz)(PPh3)] (5, Htetz = tetrazole). Selective and effective antimicrobial activities against two gram-positive bacteria (B. subtilis, S. aureus) and modest activities against one yeast (C. albicans) found in these gold(I) complexes 1-4 are noteworthy, in contrast to poor activities observed in the corresponding silver(I) complexes.

The reaction of Ni(OAc)2 with N′-[1-(2-pyridyl)ethylidene]morpholine-4-carbothiohydrazide (HL) afforded two nickel(II) complexes, [Ni(L)2] (1) and [Ni(L)(OAc)] (2), depending on reaction temperature and starting molar ratio; the former was a 6-coordinate, paramagnetic complex and the latter a 4-coordinate, diamagnetic complex. X-ray analysis of the complex 2 revealed that the ligands, L and OAc, were coordinated to the central metal ion to form a square-planar geometry. The complex 2 showed enhanced antimicrobial activities against selected Gram-positive bacteria, compared with those of the ligand alone, whereas the other complex 1 showed no activity.

The tetrameric anionic species {Na3[Ag(Htma)]4-}–(m/z= 1092.8; H3tma = thiomalic acid) is detected by negative-ion electrospray ionization (ESI) mass spectrometry as a stable constituent unit derived from an aqueous solution of the oligomeric thiomalato-silver(I) complex, recently isolated and characterized as {Na[Ag(Htma)]·0.5H2O}n(n= 24–34)1, which displays antimicrobial activity against selected bacteria, yeast and moulds.

To examine the transforming potential of the X gene product of hepatitis B virus (HBV), the X-gene-containing region (referred to as the HBx region) was introduced into mouse NIH3T3 cells. Each transformed cell line expressed X-coding mRNA at a different level. A positive correlation was found between the level of X-coding mRNA and the saturation density of the cells. The HBx-transformed cell lines exhibited X protein production and tumor formation in nude mice. The function of HBV in oncogenesis may involve the continuous expression of the X-gene-coded product in the HBV DNA-integrated cells.

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.

For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates.