Effect of Dietary Green Tea Catechin Preparation on Oxidative Stress Parameters in Large Intestinal Mucosa of Rats

Intake of green tea catechin (GTC) for 4 weeks was found to elevate vitamin E level in the mucosa of the rat large intestine. Iron-induced lipid peroxidation of the mucosal homogenate was suppressed by intake of GTC in rats fed monounsaturated fatty acid (MUFA), indicating that the protective effect of dietary GTC on mucosal oxidative stress is enhanced by combination with a MUFA-rich diet.     

Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 61.0+/-2.5, 105.0+/-4.9 and 67.0+/-3.2 microg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9+/-6.1% and 95.4+/-2.2% at the doses of 25 and 50 mg/kg, respectively. [3H]AA liberation induced by collagen in [3H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A2 (TXA2) and prostaglandin D2 (PGD2) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA2 synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA2 formation through the inhibition of AA liberation and TXA2 synthase.     

In Vivo and In Vitro Expression of Gangliosides in Chick Retina Müller Cells

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gentamicin-based medium for the isolation of group D streptococci and application of the medium to water analysis.

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH(2)PO(4), 2.0 g of NaHCO(2), 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO(3) (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Effect of green tea catechins on oxidative DNA damage of hamster pancreas and liver induced by N-Nitrosobis(2-oxopropyl)amine and/or oxidized soybean oil

It has been indicated that high fat diet is a risk factor of the pancreatic cancer by epidemiological studies. We examined whether the oxidized soybean oil (ox-oil) express the synergistic effect on the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in nuclear DNA of hamster pancreas induced by N-Nitrosobis(2-oxopropyl)amine (BOP) and whether the green tea catechins (GTC) suppressed it. Ox-oil was prepared by air oxidation, and the content of lipid hydroperoxide was 6.22 mg/ml. Hamsters were administered 0.3 ml of ox-oil/day orally for 4 weeks before BOP treatment. GTC was given ad libitum as a 0.1% aqueous solution. Four hours after subcutaneous administration of BOP, hamsters were sacrificed, and the contents of 8-oxodG were measured in nuclear DNA of pancreas and liver. The 8-oxodG content in the pancreas was increased by BOP and/or ox-oil administration. However, it was not suppressed by an intake of GTC. In the liver, though the content of 8-oxodG was increased by ox-oil, it tended to suppress the rise of 8-oxodG by a GTC intake. These results suggested that the long term intake of ox-oil might have the possibility to induce carcinogenesis in hamster pancreas and liver, and an intake of GTC might have the beneficial effect on liver.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods.

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

The trinucleotide repeat sequence d(GTC)15 adopts a hairpin conformation.

The structure of a single-stranded (ss) oligonucleotide containing (GTC)15 [ss(GTC)15] was examined. As a control, parallel studies were performed with ss(CTG)15, an oligonucleotide that forms a hairpin. Electrophoretic mobility, KMnO4 oxidation and P1 nuclease studies demonstrate that, similar to ss(CTG)15, ss(GTC)15 forms a hairpin containing base paired and/or stacked thymines in the stem. Electrophoretic mobility melting profiles performed in approximately 1 mM Na+ revealed that the melting temperature of ss(GTC)15 and ss(CTG)15 were 38 and 48 degrees C respectively. The loop regions of ss(GTC)15 and ss(CTG)15 were cleaved by single-strand-specific P1 nuclease at the T25-C29 and G26-C27 phosphodiester bonds respectively (where the loop apex of the DNAs is T28). Molecular dynamics simulations suggested that in ss(GTC)15 the loop was bent towards the major groove of the stem, apparently causing an increased exposure of the T25-C29 region to solvent. In ss(CTG)15 guanine--guanine stacking caused a separation of the G26 and C27 bases, resulting in exposure of the intervening phosphodiester to solvent. The results suggest that ss(GTC)15 and ss(CTG)15 form similar, but distinguishable, hairpin structures.     

Antiobesity effects of green tea catechins: a mechanistic review

Green tea catechins (GTC) are polyphenolic compounds present in the unfermented dried leaves of the plant, Camellia sinensis. Results from a number of randomized, controlled intervention trials have shown that consumption of GTC (270 mg to 1200 mg/day) may reduce body weight and fat. There are several proposed mechanisms whereby GTC may influence body weight and composition. The predominating hypothesis is that GTC influences sympathetic nervous system (SNS) activity, increasing energy expenditure and promoting the oxidation of fat. Caffeine, naturally present in green tea, also influences SNS activity, and may act synergistically with GTC to increase energy expenditure and fat oxidation. Other potential mechanisms include modifications in appetite, up-regulation of enzymes involved in hepatic fat oxidation, and decreased nutrient absorption. This article reviews the evidence for each of these purported mechanisms, with particular reference to studies in humans.     

Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP

Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding.     

Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT)

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.     

Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase

Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g. (CAG)(16)(CTG)(4)), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 degrees C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG approximately GAC > CTG. Loops of GTC compared with CTG melt 9 degrees C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation.     

激光微切割与定量PCR技术分析肾脏病理切片RNA

采用激光微切割与定量PCR技术,分析使用不同提取方法从不同固定方法固定的病理切片中提取的RNA.用70%乙醇、丙酮、甲醇、4%多聚甲醛固定肾脏冰冻切片,使用激光微切割技术切取肾小球,用硫氰酸胍方法(guanidinethiocyanatemethods,GTC)和Trizol试剂方法提取RNA,使用Taqman定量PCR方法分析比较各组RNA的量;选取丙酮固定的石蜡切片,使用激光微切割技术切取肾小球,采用RNA裂解液提取RNA,使用Taqman定量PCR方法,比较石蜡切片和冰冻切片中RNA含量.结果显示:提取沉淀性固定剂如乙醇、丙酮、甲醇固定的冰冻切片的RNA时,2种提取方法和3种固定方法对RNA含量的影响都无明显差异;但在提取4%多聚甲醛固定冰冻切片时,使用Trizol提取RNA含量明显高于使用GTC方法,且其含量与沉淀性固定剂固定的切片RNA含量无明显差异.石蜡切片中经激光微切割肾小球的RNA含量与冰冻切片经激光微切割肾小球的RNA含量无明显差异.结果提示:切片的固定方法和RNA的提取方法是影响切片RNA提取量的主要原因.     

Collection of airborne bacteria and yeast through water-based condensational growth

One limitation in air sampling of airborne microorganisms is their inactivation by forceful impaction and/or dehydration during the collection process. Proper inhalation risk assessments require proof of viability, as non-viable microorganisms cannot cause infectious diseases. In this study, laboratory-generated aerosols of a vegetative bacterium (E. coli) or yeast (S. kudriavzevii) were collected by a laminar-flow water-based condensational “growth tube collector (GTC),” and the GTC’s collection efficiencies were compared with those using an industry standard BioSampler. Collection efficiencies resulting from two types of collection media, phosphate-buffered saline (PBS) and nutrient media (Nutrient Broth, NB, for E. coli, and Yeast Tryptone Glucose Broth, YTGB, for S. kudriavzevii) were also assessed. Both the GTC and the BioSampler performed equally when PBS was used as the collection medium for E. coli, whereas more viable E. coli cells were collected in the GTC than the BioSampler with NB. For S. kudriavzevii, the GTC outperformed the BioSampler using either PBS or YTGB. This is likely because aerosolized E. coli cells can better survive impaction than S. kudriavzevii under the conditions used, and the BioSampler has a much higher collection efficiency for particles in the size range of single-celled E. coli than S. kudriavzevii. Moreover, the GTC had a detection limit one order of magnitude lower for yeast aerosols compared with that of the BioSampler. These results indicate that the GTC is a promising device for sampling viable aerosolized gram-negative bacteria and yeast, as it is less damaging to these types of microorganisms during the collection process.     

Cloning (CAG/GTC)n STSs by an Alu-(CAG/GTC)n PCR method: an approach to human chromosome 12 and spinocerebellar ataxia 2 (SCA2).

We report here an Alu-(CAG/GTC)n PCR method for the cloning of STSs with (CAG/GTC)n sequences. We have applied this method to genomic DNA of a somatic cell hybrid containing human chromosome 12 where linkage has been found for a known familiar dominant ataxia (SCA2), which is thought to be due to a (CAG/GTC)n expansion. We have isolated several clones containing (CAG/GTC)n sequences, which include previously identified sequences that map to chromosome 12. This method could be a new PCR approach for the cloning of repeats based on their proximity to Alu sequences.     

Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity

DNA trinucleotide repeat (TRs) expansion beyond a threshold often results in human neurodegenerative diseases. The mechanisms causing expansions remain unknown, although the tendency of TR ssDNA to self-associate into hairpins that slip along their length is widely presumed related. Here we apply single molecule FRET (smFRET) experiments and molecular dynamics simulations to determine conformational stabilities and slipping dynamics for CAG, CTG, GAC and GTC hairpins. Tetraloops are favored in CAG (89%), CTG (89%) and GTC (69%) while GAC favors triloops. We also determined that TTG interrupts near the loop in the CTG hairpin stabilize the hairpin against slipping. The different loop stabilities have implications for intermediate structures that may form when TR-containing duplex DNA opens. Opposing hairpins in the (CAG) ∙ (CTG) duplex would have matched stability whereas opposing hairpins in a (GAC) ∙ (GTC) duplex would have unmatched stability, introducing frustration in the (GAC) ∙ (GTC) opposing hairpins that could encourage their resolution to duplex DNA more rapidly than in (CAG) ∙ (CTG) structures. Given that the CAG and CTG TR can undergo large, disease-related expansion whereas the GAC and GTC sequences do not, these stability differences can inform and constrain models of expansion mechanisms of TR regions.