Theanine, gamma-glutamylethylamide, a unique amino acid in tea leaves, modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats.

Theanine (gamma-glutamylethylamide) is one of the major amino acid components in green tea and can pass through the blood-brain barrier. Recent studies suggest that theanine affects the mammalian central nervous system; however, the detailed mechanism remains unclear. In this study, we demonstrated the effect of theanine on neurotransmission in the brain striatum by in vivo brain microdialysis. Theanine injection into the rat brain striatum did not increase the concentration of excitatory neurotransmitters in the perfusate. On the other hand, theanine injection increased the concentration of glycine in the perfusate. Because it has been reported that theanine promotes dopamine release in the rat striatum, we investigated the glycine and dopamine concentrations in the perfusate. Co-injection of glycine receptor antagonist, strychnine, reduced theanine-induced changes in dopamine. Moreover, AMPA receptor antagonist, which regulates glycine and GABA release from glia cells, inhibited these effects of theanine and this result was in agreement with the known inhibitory effect of theanine at AMPA receptors.

Amla (Emblica officinalis Gaertn.) extracts reduce oxidative stress in streptozotocin-induced diabetic rats.

The antioxidant properties of amla extracts and their effects on the oxidative stress in streptozotocin-induced diabetes were examined in rats. Amla in the form of either the commercial enzymatic extract SunAmla (Taiyo Kagaku Co. Ltd., Yokkaichi, Japan) (20 or 40 mg/kg of body weight/day) or a polyphenol-rich fraction of ethyl acetate extract (10 or 20 mg/kg of body weight/day) was given orally for 20 days to the streptozotocin-induced diabetic rats. Amla extracts showed strong free radical scavenging activity. Amla also showed strong inhibition of the production of advanced glycosylated end products. The oral administration of amla extracts to the diabetic rats slightly improved body weight gain and also significantly alleviated various oxidative stress indices of the serum of the diabetic rats. The elevated serum levels of 5-hydroxymethylfurfural, which is a glycosylated protein that is an indicator of oxidative stress, were significantly reduced dose-dependently in the diabetic rats fed amla. Similarly, the serum level of creatinine, yet another oxidative stress parameter, was also reduced. Furthermore, thiobarbituric acid-reactive substances levels were significantly reduced with amla, indicating a reduction in lipid peroxidation. In addition, the decreased albumin levels in the diabetic rats were significantly improved with amla. Amla also significantly improved the serum adiponectin levels. These results form the scientific basis supporting the efficacy of amla for relieving the oxidative stress and improving glucose metabolism in diabetes.

Polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection.

Synthetic sialic acid-containing macromolecules inhibit influenza virus attachment to target cells and suppress the virus-mediated hemagglutination and neutralize virus infectivity in cell culture. To test the protective effects of attachment inhibitors in vivo, mice were infected with mouse-adapted influenza virus A/Aichi/2/68 (H3N2) and treated with synthetic polyacrylamide-based sialylglycopolymer PAA-YDS bearing moieties of (Neu5Acalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1)2-3,6Manbeta1-4GlcNAcbeta1-4GlcNAc. Single intranasal inoculations with PAA-YDS 30 min before or 10 min after infection increased the survival of mice (P<0.01). Multiple treatments with aerosolized PAA-YDS on days 2-5 post infection also increased survival (P<0.01), alleviated disease symptoms, and decreased lesions in the mouse lungs. These data suggest that synthetic polyvalent inhibitors of virus attachment can be used for prevention and treatment of influenza.

Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea.

BACKGROUND: Hyperimmunized bovine colostrum containing antibodies has been shown to be effective in the treatment of rotavirus diarrhea. Antibodies derived from eggs of immunized hens may be a less expensive and more practical alternative. In this study, children with proven rotavirus diarrhea were treated with immunoglobulin extracted from eggs of chicken immunized with human rotavirus strains. METHODS: In a randomized, double-blind study, 79 children with known rotavirus diarrhea were assigned to receive either 10 g hyperimmune egg yolk (HEY) daily in four equally divided doses for 4 days (HEY group) or a similar preparation obtained from nonimmunized chicken (placebo group). The daily stool frequency and amount, oral rehydration solution iORS) intake, and presence of rotavirus in the stool were monitored for 4 days. RESULTS: In the HEY-treated group, there was significant reduction in stool output (in grams per kilogram per day; HEY vs. placebo; 87+/-59 vs. 120+/-75, P = 0.03), and significant reduction of ORS intake (in milliliters per kilogram per day) (HEY vs. placebo; 84+/-46 vs. 122+/-72, P = 0.008) on day 1 and clearance of virus on day 4 (HEY vs. placebo; 73% vs. 46%, P = 0.02). There was, however, no difference in diarrheal duration between the groups. CONCLUSIONS: Treatment with HEY against four human rotavirus strains resulted in modest improvement of diarrhea associated with earlier clearance of rotavirus from stools. These results indicate an encouraging role of HEY in the treatment of rotavirus-induced diarrhea in children. Further studies are needed to optimize the dose and neutralization titer and thus improve the efficacy of egg yolk immunoglobulin IgY derived from immunized hens.

Preventive effect of partially hydrolyzed guar gum on infection of Salmonella enteritidis in young and laying hens.

The preventive effect of partially hydrolyzed guar gum (PHGG) on the colonization of Salmonella enteritidis (SE) in young and laying hens was investigated. The effects of feed supplemented with 0.025, 0.05, and 0.1% PHGG was examined on young hens orally infected with SE. The incidence of SE in organs was decreased, the excretion of SE into feces was increased, and the agglutinating antibody titer to SE in serum was decreased by the administration of PHGG to young hens. In particular, feed supplemented with 0.025% PHGG was the most effective. It was also shown that feed supplemented with 0.025% PHGG increased the number of Bifidobacterium spp. and Lactobacillus spp., the most numerous intestinal bacteria in the cecum of young hen. The effect of the excretion of SE via feces was also observed in an experiment using laying hens. The incidence of SE on the surface of the eggshell and in egg white and egg yolk was also decreased when the feed of laying hens was supplemented with 0.025% PHGG. These results show that the administration of feed supplemented with PHGG can prevent the colonization of SE in young and laying hens, which, in turn, could be related to improvement in the balance of intestinal microflora.

Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs.

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.

Antimicrobial effects of green tea polyphenols on thermophilic spore-forming bacteria.

The inhibitory action of tea polyphenols towards the development and growth of bacterial spores was examined. Among the tested Bacillus bacteria, tea polyphenols showed antibacterial effects towards Bacillus stearothermophilus, which is a thermophilic spore-forming bacterium. The heat resistance of B. stearothermophilus spores was reduced by the addition of tea polyphenols. Clostridium thermoaceticum, an anaerobic spore-forming bacterium, also exhibited reduced heat resistance of its spores in the presence of tea polyphenols. (-)-Epigallocatechin gallate, which is the main component of tea polyphenols, showed strong activity against both B. stearothermophilus and C. thermoaceticum. The heat resistance of these bacterial spores was more rapidly decreased by the addition of tea polyphenols at high temperatures.

Tea catechin supplementation increases antioxidant capacity and prevents phospholipid hydroperoxidation in plasma of humans.

The effect of green tea catechin supplementation on antioxidant capacity of human plasma was investigated. Eighteen healthy male volunteers who orally ingested green tea extract (254 mg of total catechins/subject) showed 267 pmol of epigallocatechin-3-gallate (EGCg) per milliliter of plasma at 60 min after administration. The plasma phosphatidylcholine hydroperoxide (PCOOH) levels attenuated from 73.7 pmol/mL in the control to 44.6 pmol/mL in catechin-treated subjects, being correlated inversely with the increase in plasma EGCg level. The results suggested that drinking green tea contributes to prevent cardiovascular disease by increasing plasma antioxidant capacity in humans.

Comparison of egg-yolk protein hydrolysate and soyabean protein hydrolysate in terms of nitrogen utilization.

Egg-yolk protein hydrolysate (YPp) is an alternative protein source in formulas for infants with intolerance to cow's milk or soyabean protein, or for patients with intestinal disorders. However, the nutritional value of YPp has never been investigated. YPp was prepared by enzymic hydrolysis of delipidated yolk protein, which led to an average peptide length of 2.6 residues. Three experiments were performed. In Expt 1, we compared the intestinal absorption rate of YPp and soyabean protein hydrolysate (SPp) in rats. YPp and SPp solutions were injected into the duodenum of anaesthetized rats and blood samples were taken from the portal vein at 7, 15, 30, 60, and 120 min. A higher amino acid concentration in the serum of the YPp group demonstrated that YPp was absorbed faster than SPp. In Expt 2, the effects of dietary YPp and SPp on body-weight gain, protein efficiency ratio (PER) and feed efficiency ratio (FER) were determined. At the end of the experiment, body weight had increased in both groups, while PER and FER were significantly higher in rats fed on YPp. In Expt 3, to investigate the effects of dietary YPp and SPp on N metabolism, we determined the biological value and net protein utilization. Yolk protein was the reference protein. Biological value and net protein utilization values were very similar between animals fed on yolk protein and YPp diets, and significantly higher than in rats fed on the SPp diet. The present findings demonstrate that there is no adverse effect of hydrolysis of yolk protein on N utilization, and that the nutritive value of YPp is similar to that of yolk protein and superior to that of SPp.

A novel method for determination of alpha1,6fucosyltransferase activity using a reducing oligosaccharide from egg yolk as a specific acceptor.

A new method for determination of alpha1,6fucosyltransferase activity has been described. Recently, the disialyl-biantennary undecasaccharide was prepared in high yield from egg yolk [(1996), Carbohydr Lett 2: 137-42]. By treatment of this oligosaccharide with neuraminidase and beta-galactosidase, we readily obtained an asialo-agalacto-biantennary heptasaccharide (GlcNAcbeta 1,2Manalpha1,6[GlcNAcbeta1,2Manalpha1,3]Manbeta1 ,4GlcNAcbeta1,4GlcNAc). Using this asialo-agalacto-oligosaccharide as an acceptor, fucosyltransferases from human plasma and extracts of various human hepatoma cell lines were assayed in the presence of GDP-[3H]fucose. The reaction mixture was applied to a column of GlcNAc-binding, Psathyrella velutina lectin coupled gel. All the fucosylated acceptor were bound to the column which was eluted with 50 mM GlcNAc. Structural analyses revealed that only the innermost GlcNAc residue of the acceptor was fucosylated through an alpha1,6-linkage, and the oligosaccharide prepared could be used as a specific acceptor for alpha1,6fucosyltransferase. The present method was used to screen plasma alpha1,6fucosyltransferase in several patient groups, and significantly elevated activities were found in samples from patients with liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection.

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 microM to 40 microM). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 microM in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent.

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.

Occurence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.

Simultaneous determination of catechins in human saliva by high-performance liquid chromatography.

Green tea extracts have been suggested to possess a preventive effect against dental caries. A quantitative method for their anticariogenic substances, catechins, was developed to evaluate their concentrations in human saliva after mouthrinsing with green tea extract. Salivary catechins were extracted to the organic phase after forming a complex with diphenylborate and an ion-pair with tetra-n-butylammonium, and then back-extracted to the acidic aqueous phase. The extract was analyzed by high-performance liquid chromatography using diode array detection at absorption wavelengths ranging from 269 to 278 nm. In reversed-phase chromatography by a gradient elution, eight catechins originating from green tea and an internal standard were separated in 15 min without interfering peaks. All the catechins were simultaneously and selectively determined in the concentration range 0.05-25.0 microg/ml. In replicate spiking experiments with standards, the mean recovery ranged between 86 and 99%, and both intra- and inter-assay C.V.s were within 2.3%. When mouthrinsing with an aqueous solution of green tea extract (5.0 mg/ml) containing eight catechins, the quantitative results revealed that each catechin was retained at microg/ml levels in saliva for up to 60 min.

Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin.

A dimeric sialyl Lewis X (SLex) glycopeptide was synthesized enzymatically in three steps from an N-linked oligosaccharide prepared from egg yolk. Treatment of delipidated hen egg yolk with the protease Orientase and neuraminidase gave a dimeric N-acetyllactosamine-containing oligosaccharide linked to asparagine. Addition of sialic acid and fucose catalyzed by alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase provided the dimeric SLex, which was shown to be as active as monomeric SLex as an inhibitor of E-selectin with IC50 0.75 mM. The synthetic dimeric SLex of the mucin type (i.e. SLex linked to the 3- and 6-OH groups of Gal) is, however, about five times as active as the monomer. It is suggested that dimeric SLex glycopeptides of the mucin type would be effective ligands for E-selectin.

Preparation of N-acetylneuraminic acid from delipidated egg yolk.

Egg yolk, a large proportion of the egg, was studied for the preparation of N-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 degrees C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 microS cm-1. The filtrate was applied on a column of Dowex HCR-W2 (20-50 mesh), followed by a column of Dowex 1-X8 (200-400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO2H (0-2 M). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 degrees C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was > 98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.

Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources.

Transphosphatidylation from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was conducted by seven phospholipase D preparations from different sources, one of which was of cabbage origin and other six of Streptomyces origin. The reactions were carried out at 30 degrees C using mixture of ethyl acetate containing PC and buffer containing ethanolamine and phospholipase D. To obtain the activity ratio of transphosphatidylation and hydrolysis at various ratios of ethanolamine and water concentrations, the apparent rate constant ratios of transphosphatidylation and hydrolysis, (k'3/k3)app, were calculated, keeping the concentration of PC constant (17.8 mM). Among the seven enzymes examined, five showed good transphosphatidylation, in which 100% of PC could be converted to PE at ethanolamine concentrations ranging from 0.3 to 1.0 M, while two showed poor transphosphatidylation activity. At higher ethanolamine concentrations, the reaction rate was decreased due to substrate inhibition. Hydrolytic reactions were conducted at 30 degrees C with respective enzymes by using mixture of ethyl acetate containing PC or PE and buffer containing phospholipase D. The ratio of (kcat/Km)PE/(kcat/Km)PC was calculated to determine the substrate specificity of various phospholipase D enzymes. The values of (k'3/k3)app varied with the origin of the enzymes, whereas the values of (kcat/Km)PE/(kcat/Km)PC were not so different. The results obtained show that (k'3/k3)app is a good parameter to select an enzyme and that the timing of stopping the reaction is also important to avoid the hydrolysis of the product.