Ginsenoside Re promotes human sperm capacitation through nitric oxide-dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

Re(V) complexes formed by metal-assisted solvolysis of di-(2-pyridyl)ketone: Synthesis, X-ray studies, redox behavior and DFT calculations

The complexes {ReOCl₂[(py)₂C(O)(OR)]}, (R = CH₂CH₃ (1), CH₂CH₂CH₃ (2)) were obtained from the metal-assisted alcoholysis reaction of di-2-pyridylketone. Their crystal structures were determined by single-crystal X-ray diffraction. The structures of 1 and 2 consist of neutral mononuclear molecules containing the [ReO]³⁺ core. In both compounds the Re(V) central ion is also bonded to an oxygen and both nitrogens from the organic ligand and two chlorides in a distorted octahedral environment. Cyclic voltammograms in CH₃CN solutions showed two main redox responses, both of them involving one electron transfer. Comparative theoretical studies on equilibrium geometries and electronic properties were conducted in the framework of the density functional theory (DFT).     

Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.     

OPTIMIZATION OF DNA EXTRACTION FROM BROWN ALGAE (PHAEOPHYCEAE) BASED ON A COMMERCIAL KIT1

Large‐scale DNA molecular studies require reliable and efficient tools for DNA extractions. However, for some plant species and brown algae, isolation of high‐quality DNA is difficult. We developed a novel method for isolating high‐quality DNA from the polysaccharide‐rich and polyphenol‐rich brown algae based on a commercial kit and protocol (Qiagen) by optimizing the lysis step and including a chloroform/isoamyl alcohol supplementary purification step. DNAs from 24 brown algal species extracted using the original and the modified Qiagen protocol were compared for yield, quality, and effectiveness in PCR amplification. There was no significant difference in the yields between protocols. However, a statistically significant increase in DNA purity was obtained with the modified protocol, for which the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ absorbance ratios averaged 1.66 ± 0.05 and 1.31 ± 0.01, respectively, compared to 1.37 ± 0.04 and 0.52 ± 0.04 with the original protocol. DNAs extracted by the modified procedure were more successfully amplified by PCR (nuclear, mitochondrial, and chloroplastic regions) than DNAs extracted using the original commercial kit and protocol. Importantly, the modified protocol can be applied in a high‐throughput (e.g., 96‐well plate) format, allowing a higher efficiency for downstream molecular analysis. In addition, improved DNA quality could increase its stability for long‐term storage.     

Removal of nonionic surfactants from wastewater using a constructed wetland

Removal of nonionic surfactants from municipal wastewater using a constructed wetland with a horizontal subsurface flow was studied in 2009 and 2010. Extraction spectrophotometry with 3′,3″,5′,5″‐tetrabromophenolphthalein ethyl ester and KCl served to determine the analyte concentrations. Triton^® X‐100 was used as a standard to express the nonionic‐surfactant concentrations. Anionic and cationic surfactants were shown not to interfere during the determination. Nonionic surfactants were degraded (to products undeterminable by the method) with a high average efficiency that reached 98.1% in 2009 and 99.1% in 2010, respectively. The average concentration of nonionic surfactants at the inflow was 0.978 mg/l, while it was close to the limit of quantification at the outflow (0.014 mg/l). A significant fraction of nonionic surfactants (38.7%) was already degraded during the pretreatment, and only 14.0% of the nonionic surfactants remained in the interstitial H₂O taken in the vegetation bed at a distance of 1 m from the inflow zone at a 50‐cm depth. Nonionic surfactants were degraded both under aerobic and anaerobic conditions.     

Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation

Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.