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Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/ in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms. [ABSTRACT FROM AUTHOR]

Zinc-finger nucleases ( ZFNs) are engineered nucleases that induce DNA double-strand breaks ( DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining ( NHEJ) or by homologous recombination ( HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP- ZFN mRNAs into the embryos of an EGFP-transgenic line and observed the mutation frequency in the target site of EGFP. We also examined the effects of the EGFP- ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis. [ABSTRACT FROM AUTHOR]

Zinc-finger nucleases ( ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka ( Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka. [ABSTRACT FROM AUTHOR]

We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC ( HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo. [ABSTRACT FROM AUTHOR]

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants. Developmental Dynamics 238:2511-2521, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In cases of essential fatty acid deficiency, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid as a 20-carbon analog of arachidonic acid. It was reported that 20:3n-9 levels were markedly higher in human fetal cartilage than in the muscle, liver and spleen. We, therefore, hypothesized that 20:3n-9 decreased osteoblastic activity. Goldfish scales were incubated either with 20:3n-9 or with oleic acid at 15 °C for 6 and 18 h. Both osteoblastic and osteoclastic activities in the scale were assessed by measuring alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase, respectively. MC3T3-E1 cells (an osteoblast cell line derived from the mouse) were incubated with 20:3n-9 or oleic acid at 37 °C for 6 and 18 h. ALP activity in cell lysate was measured. In the case of experiments with scales, 20:3n-9 (1–100 μM) significantly suppressed osteoblastic activity after 6 and 18 h of incubation, whereas oleic acid did not change this activity. Osteoclastic activity was not affected either by 20:3n-9 or by oleic acid. In the case with the cell line, osteoblastic activity was again significantly decreased with 20:3n-9 (10–30 μM) after 6-h incubation but not after 18 h incubation. The presence of 20:3n-9 in fetal cartilage may be important for the prevention of calcification in the cartilage. 20:3n-9 could be applied to some clinical situations where bone formation should be inhibited. [ABSTRACT FROM AUTHOR]

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter- GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo. Developmental Dynamics 237:2475-2482, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its ‘silencing’, occurred in parallel in the nucleus. [Copyright &y& Elsevier]

The objectives of this study were to evaluate the effects of traditional herbal medicine on elderly patients with weakness, and to devise a suitable study design for assessing the clinical effectiveness of traditional herbal medicines. Twenty-one elderly patients with weakness (mean age, 78.2 ± 7.5; male : female, 8 : 13) were studied using a self-controlled design with a run-in period. The observation term was 3 months, and quality of life (short form-36 and profile of mood status) were adopted as evaluation endpoints. In addition, natural killing activity and surface antigens (CD19, CD3, CD4, CD16, CD56, CD158a, CD158b) on lymphocytes obtained from peripheral blood were analyzed to evaluate patients’ immune status. EK-41 (Hochu-ekki-to), EK-48 (Juzen-taiho-to) and EK-98 (Ogi-kenchu-to) were administered to 10, 10 and one patients, respectively. There were no dropouts due to side-effects. Results of the short form-36 were significantly improved after 3 months, with the patients in the EK-48 group showing greater improvement than those in the EK-41 group. Each component of the profile of mood status was improved by the treatment, and the improvement of V, D and F was especially significant. D and A-H were considerably improved in the patients of the EK-48 group. In contrast, the improvement of D and T-A was most marked in the EK-41 group. Some augmentation of NK activity was observed after 3 months, but the effect was not significant. Although neither the CD4/8 ratio nor the percentages of CD3+, CD19+ or CD16+ CD158a + cells was changed significantly, the percentage of CD56+, CD56+ CD16+ and CD16+ CD158b + cells were significantly increased. A preliminary clinical trial for elderly patients with weakness was carried out to assess the efficacy of traditional medicines, resulting in evidence of their clinical and immunomodulating effects, although this evidence had some limitations. In addition, we obtained some insights into the elements of designing studies suitable for assessing the clinical efficacy of traditional herbal medicines. [ABSTRACT FROM AUTHOR]

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein α2 and β subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Isoferulic acid (IFA) is a main active ingredient of the rhizoma of Cimicifuga heracleifolia, which is used frequently in Japanese traditional medicine as an anti-inflammatory drug. It has been revealed that IFA inhibits the production of macrophage inflammatory protein-2 (MIP-2), which is a murine counterpart of the chemokine family that may contribute to the pathogenesis of inflammatory diseases through the chemotactic activity for inflammatory and immune effector cells. Aim of the study: In this study, we investigated the therapeutic effect of IFA on the progression of lethal influenza virus pneumonia in mice by comparison with that of dexamethasone (DX), a potent inhibitor for various inflammatory cytokines including MIP-2. Methods: Mice were infected by intranasal inoculation of influenza virus under ether anesthesia. The IFA or DX was given by oral administration once daily for 4 days after infection. After infection, the survival rate and the change in body weight were daily monitored. Results: IFA administration markedly improved the survival rate and body weight loss of influenza virusinfected mice in a suitable dose range (0.5 mg/day). However, DX administration did not show a beneficial effect at any dose. Conclusion: These data suggested that IFA is a novel tool not only for the intervention therapy, but also for the studies on the pathogenesis of influenza virusinduced pneumonia. [ABSTRACT FROM AUTHOR]

Explores the cloning of cDNA sequences encoding functional Delta5 fatty acid desaturase genes from Dictyostelium discoideum yeast fungi. Amino-acid sequence of cDNA; Substrate specificity of Delta5 desaturates.

Evaluates delta 5-fatty acid desaturase identification from the cellular mold Dictyostelium discoideum. Alignment of the deduced amino acid sequence; Indication of the predicted hydrophobicity plot for desaturase; Amplification of genomic DNA.

Although it is now accepted that killer-cell inhibitory receptors (KIRs), which were molecularly cloned in 1995, deliver negative signals to natural killer (NK) cells regarding the recognition of target cells, it is still unclear how the expression of these receptors on lymphocytes is regulated. Therefore, we investigated the regulation of expression of representative KIRs, CD158a and CD158b, by cytokines such as interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Neither IL-4 nor IFN-gamma affected the expression of CD158a/b, but incubation for 48 h with IL-2, which enhances the killer activity of NK cells, upregulated the expression of the KIRs. This upregulation by IL-2 was also observed in CD16-positive cells sorted from total lymphocytes. In contrast, IL-4, which is a downregulator of IL-2-induced killer responses, did not change the level of CD158a/b expression when added after the IL-2 treatment. These findings suggest that IL-2 plays an important role in the regulation of CD158a/b expression, and might be involved in controlling NK activity via regulating expression of these molecules. [ABSTRACT FROM AUTHOR]

The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein, This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the ease. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base. The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol. [ABSTRACT FROM AUTHOR]

The cellular slime mold Polysphondylium pallidum was grown upon Escherichia coli B/r, and the fatty acid compositions of total lipids obtained from vegetative amebae and aggregation-competent cells were compared. Fatty acids isolated from vegetative cells included C-17 and C-19 cyclopropane fatty acids and also straight-chain, saturated fatty acids. The cyclopropane fatty acids were derived from the ingested bacteria. Development of amebae to aggregation-competent cells was accompanied by a substantial decrease in saturated cyclopropane fatty acids and a concomitant increase in unsaturated fatty acids and unsaturated cyclopropane fatty acids, mostly as 18∶3 (5,9,12). We report here the fatty acid composition and identify the occurrence of Δ5 desaturation of cyclopropane fatty acids, namely, 9,10-methylene 5-hexadecenoic acid and 11,12-methylene 5-octadecenoic acid. These fatty acids have not been reported previously in the related species Dictyostelium discoideum, which also feeds on E. coli B/r and has Δ5-desaturation activity. [ABSTRACT FROM AUTHOR]

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysyl-endopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine- linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins. [ABSTRACT FROM AUTHOR]

Protein compositions of plasma membranes of Dictyostelium discoideum and their developmental changes during aggregation were analyzed by two-dimensional polyacrylamide gel electrophoresis. More than 150 polypeptide species could be detected with the growth-phase cells by staining. Most of them (about 120 spots) were conserved during aggregation, although their relative intensities of staining changes. On the initiation of cell differentiation, new polypeptides were accumulated on the plasma membrane and at least 100 new polypeptides were detected at the late-aggregation stage. This accumulation of new components was completely inhibited when the growth-phase cells were incubated in a liquid medium containing 5 mM EDTA. Studies on protein synthesis suggested that the accumulation and appearance of the new protein components was due to de novo synthesis. Pulse-label experiments with [35S]methionine showed that out of 200 proteins detected during aggregation, about 90 proteins showed various stage-specific patterns of synthesis and the rest were synthesized consistently. Actin and discoidin present in the plasma membrane were identified by reference to purified actin and discoidin. Actin synthesized de novo accumulated progressively on the plasma membrane during aggregation. Discoidin also accumulated on the plasma membrane, but a large amount was also recovered in a cytoplasmic soluble fraction. Discoidin I was separated into two spots with different pI values on a two-dimensional gel. [ABSTRACT FROM AUTHOR]

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium metaperiodate oxidation and sodium boro[³H]hydride reduction. The amount of incorporation of tritium from NaB³H4 reached a plateau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density-gradient centrifugation showed that the plasma membranes were selectively labeled by this technique. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100 °C. The released materials were eluted at a bed volume after chromatography using a Sephadex G-50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides. Using the above method, the plasma membranes from aggregation-phase cells were labeled four times more than those from growth-phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one-dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase. Using this method, the surface glycoproteins can be analyzed directly by gel electrophoresis of the lysate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 12-year-old boy with chronic active Epstein-Barr virus infection (CAEBV) and has developed fatal acute circulatory failure during the preconditioning of hematopoietic stem cell transplantation (HSCT). Laboratory test find that degeneration of cardiac muscle and large vessel arteritis associated with CAEBV through an autopsy of the patient. The findings suggest that hemophagocytic syndrome and degeneration of cardiac muscles will cause the fatal arrhythmia.

Background: Influenza virus is a worldwide health problem with significant economic consequences. To study the gene expression pattern induced by influenza virus infection, it is useful to reveal the pathogenesis of influenza virus infection; but this has not been well examined, especially in vivo study. Aims: To assess the influence of influenza virus infection on gene expression in mice, mRNA levels in the lung and tracheal tissue 48 h after infection were investigated by cDNA array analysis. Methods: Four-week-old outbred, specific pathogen free strain, ICR female mice were infected by intra-nasal inoculation of a virus solution under ether anesthesia. The mice were sacrificed 48 h after infection and the tracheas and lungs were removed. To determine gene expression, the membrane-based microtechnique with an Atlas cDNA expression array (mouse 1.2 array II) was performed in accordance with the manual provided. Results and conclusions: We focused on the expression of 46 mRNAs for cell surface antigens. Of these 46 mRNAs that we examined, four (CD1d2 antigen, CD39 antigen-like 1, CD39 antigen-like 3, CD68 antigen) were up-regulated and one (CD36 antigen) was down-regulated. Although further studies are required, these data suggest that these molecules play an important role in influenza virus infection, especially the phase before specific immunity. [ABSTRACT FROM AUTHOR]

The all- cis-5,9,12-heptadecatrienoic acid was identified in the cellular slime mold Polysphondylium pallidum. The structural elucidation was accomplished by capillary gas chromatography, argentation thin-layer chromatography, and gas chromatography/mass spectrometry. This fatty acid has not been reported previously. [ABSTRACT FROM AUTHOR]

When Dictyostelium cells starve, they express genes necessary for aggregation. Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation and that forms small plaques on bacterial lawns, thus indicating slow growth. Sequencing of the mutated locus showed a strong similarity to the catalytic domain of cdc2-related kinase genes. Phylogenetic analysis further indicated that the amino acid sequence was more close to cyclin-dependent kinase 8 than to the sequence of other cyclin-dependent kinases. Thus, we designated this gene as Ddcdk8 . The Ddcdk8 -null cells do not aggregate and grow somewhat more slowly than parental cells when being shaken in axenic medium or laid on bacterial plates. To confirm whether these defective phenotypes were caused by disruption of this gene, the Ddcdk8 -null cells were complemented with DdCdk8 protein expressed from an endogenous promoter, but not an actin promoter, and when the complemented cells were then allowed to grow on a bacterial lawn, they began to aggregate as the food supply was depleted and finally became fruiting bodies. The results suggest that properly regulated DdCdk8 activity is essential for aggregation. Because, when starved, Ddcdk8 -null cells do not express the acaA transcripts required for aggregation, we deduce that Ddcdk8 is epistatic for acaA expression, indicating that the DdCdk8 products may regulate expression of acaA and/or other genes. [ABSTRACT FROM AUTHOR]

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.

It has been previously reported that green-tea extract (GTE) inhibits the growth of influenza virus by preventing its adsorption. In this study, we further investigated whether GTE exerts an additional inhibitory effect on the acidification of intracellular compartments such as endosomes and lysosomes (referred to as ELS) and thereby inhibits the growth of influenza A and B viruses in Madin-Darby canine kidney cells. The vital fluorescence microscopic study showed that GTE inhibited acidification of ELS in a concentration-dependent manner. Moreover, the growth of influenza A and B viruses was equally inhibited when the cells were treated with GTE within as early as 5 to 15 min after infection, depending on the virus strains. The fact that (-)epigallocatechin (EGC), one of major catechin molecules in GTE, exerts the inhibitory effects on the acidification of ELS and virus growth in a manner similar to that of GTE strongly suggests that EGC is one of the active components in the extract.