Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

太子参粗提物抗氧化性及护肤功效分析

比较三种不同萃取方式对太子参Pseudostellaria heterophylla粗提物抗氧化、美白、保湿等方面的影响。结果显示,在ABTS和DPPH自由基及细胞内活性氧(ROS)抑制率分析中,以水萃方式所得粗提物的抑制效果最佳,优于醇萃取及超临界流体萃取。在美白功效方面,B16细胞黑色素含量抑制率以醇萃物最佳,酪氨酸酶活性抑制率则以水萃物最佳。进一步萃取太子参粗多糖体分析其保湿及吸湿效果,结果显示太子参粗多糖体具有良好的保湿及吸湿功效,其效果与透明质酸相近,应用于皮肤水分散失率评估,也具有显著的预防功效。     

Cellular proteins which can specifically associate with simian virus 40 small t antigen.

When crude, radiolabeled extracts of various cells were applied to homogeneous simian virus 40 small t antigen-Sepharose adsorbents, three cell proteins (57, 32, and 20 kilodaltons [kDa]) bound specifically. Each also bound to an insoluble, truncated t derivative composed of the COOH-terminal 123 residues of the protein. The binding of these proteins was greatly inhibited after reduction and alkylation of the t ligand. Therefore, some element of native conformation, but not all of the primary structure of t, is necessary for this binding property, which may constitute a discrete, in vitro biochemical function of this protein. Results of cell fractionation experiments suggested that the 57- and 32-kDa proteins are nonnuclear cell constituents, whereas the 20-kDa protein was closely associated with a detergent-washed nuclear fraction. Specific immunoblotting and comparative partial proteolytic digestion analyses indicated that the 57-kDa protein is tubulin, a major component of the cytoskeleton. In this regard, t and tubulin were observed to coimmunoprecipitate from crude cell extracts after incubation with monospecific anti-t antibody. Therefore, it is possible that t and tubulin interact in vivo.     

Glycolipid-lectin interactions: detection by direct binding of 125I-lectins to thin layer chromatograms

Glycolipids that bind 125I-labeled lectins are detected by autoradiography after thin layer chromatography of glycolipid standards or crude lipid extracts. Soybean agglutinin, Bandeiraea simplicifolia I isolectins A4 and B4, and Helix pomatia lectin are used to detect corresponding cell surface, glycolipid receptors in human and bovine erythrocytes. When lipid extracts from A and AB erythrocyte stroma are analyzed with Helix pomatia lectin, a polymorphic expression of blood group A glycolipid determinants is detected. The Bandeiraea simplicifolia isolectins react weakly with human erythrocyte glycolipids but bind at least 4 glycolipids in bovine stroma extracts. Soybean agglutinin reacts with glycolipids in all erythrocytes analyzed. This technique extends lectin specificity studies from inhibition analyses in aqueous systems using available, known structures to identification of specific, lectin-binding glycolipids in crude lipid extracts of cell membranes.     

Effect of simulated gastrointestinal conditions and epithelial transport on extracts of green tea and sage

Few in vitro screening studies on the biological activities of plant extracts that are intended for oral administration consider the effect of the gastrointestinal system. This study investigated this aspect on extracts of Camellia sinensis (green tea) and Salvia officinalis (sage) using antimicrobial activity as a model for demonstration. Both the crude extracts and their products after exposure to simulated gastric fluid (SGF) as well as simulated intestinal fluid (SIF) were screened for antimicrobial activity. The chromatographic profiles of the crude plant extracts and their SGF as well as SIF products were recorded and compared qualitatively by means of high performance liquid chromatography coupled to mass spectrometry. The effect of epithelial transport on the crude plant extracts was determined by applying them to an in vitro intestinal epithelial model (Caco-2). The crude extracts for both plants exhibited reduced antimicrobial activity after exposure to SGF, while no antimicrobial activity was detected after exposure to SIF. These results suggested chemical modification or degradation of the antimicrobial compounds when exposed to gastrointestinal conditions. This was confirmed by a reduction of the peak areas on the LC–UV–MS chromatograms. From the chromatographic profiles obtained during the transport study, it is evident that some compounds in the crude plant extracts were either not transported across the cell monolayer or they were metabolised during passage through the cells. It can be deduced that the gastrointestinal environment and epithelial transport process can dramatically affect the chromatographic profiles and biological activity of orally ingested natural products.     

The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

Occurrence of Only One Form of Glutamine Synthetase in the Green Alga Monoraphidium braunii

Anion-exchange chromatography of crude extracts from the green alga Monoraphidium braunii yielded two glutamine synthetase (GS) activities. The ratio of activities was markedly different when crude extracts were subjected to various processing conditions but was not influenced by environmental factors of cell cultures. However, high performance liquid chromatography anion-exchange chromatograms showed only one GS if the crude extracts were processed immediately after cell disruption. Moreover, standard chromatography of crude extracts obtained in the absence of dithioerythritol, a reductant generally used in disruption buffers, yielded a single activity peak. Enzyme samples from the two activities obtained in the presence of dithioerythritol were purified for physicochemical characterization and antibody production. Both enzyme samples exhibited similar reactions to different inactivating agents and were undistinguishable by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Additionally, the two GS preparations showed absolute antigenic identity as demonstrated by immunodiffusion and immunoblotting experiments. Immunocytochemistry of M. braunii cryosections evidenced a chloroplast-specific distribution of the enzyme, which rules out the existence of a cytoplasmic counterpart. All these results support the proposal that M. braunii possesses only one form of GS.     

Rice callus extracts for enhancing skin wound healing

Enhanced cell migration in the course of wound healing is required to repair damaged skin. We investigated the effects of rice callus extracts on the migration of skin keratinocytes. Rice callus extracts were obtained by using three different methods: pressurized hot water, crude ethanol, and liquid-liquid extractions. The extract obtained by using crude ethanol extraction was more effective in the migration of skin cells than that obtained by pressurized hot water extraction (PHWE). The crude ethanol extract (CEE) was further partitioned by performing liquid-liquid extraction. As phenolics are inhibitory compounds affecting cell migration, we analyzed the total phenolic content of the rice callus extracts. The level of phenolics in the n-hexane partitioned extract (n-HPE) of CEE was lower than that in all other partitioned extracts. The n-HPE was most effective in enhancing cell migration. We analyzed wound healingrelated factors including platelet derived growth factor B (PDGF-B), transforming growth factor beta 1 (TGF-β1), heparin-binding epidermal growth factor (HB-EGF), and fibroblast growth factor 2 (FGF-2) after the treatment of n-HPE. Most of the expressions of cell migration-related growth factors increased, but HB-EGF dramatically increased (6.5-fold) in keratinocytes treated with n-HPE. The results indicate that n-HPE contains more stimulating growth factors or proteins and less cell migration inhibiting factors than the other tested extracts; thus, n-HPE treatment produced the greatest enhancement of cell migration.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

小桐子提取物除草活性的生物测定

为全面了解小桐子(Jatropha curcas L. )提取物的除草活性,以萝卜(Raphanus sativus L. )、苋(Amaranthus tricolor L. )、苏丹草[Sorghum sudanense (Piper) Stapf]和黑麦草(Lolium perenne L. )为实验材料,对小桐子果壳和枝叶的水、乙醇(体积分数95%)、正丁醇、乙酸乙酯、氯仿和石油醚粗提物的除草活性进行了生物测定,并从中筛选出抑制作用最强的水粗提物进行进一步的活性组分分离及其除草活性的生物测定.测定结果显示,小桐子果壳和枝叶的6种溶剂提取物(10 g·L~(-1))对供试的4种植物幼苗的根长和茎高均有不同程度的抑制作用,其中水粗提物和乙醇(体积分数95%)粗提物的抑制作用较强,且水粗提物对供试的4种植物幼苗的根长和茎高的抑制作用均在75%以上,显著高于其他溶剂粗提物(P<0.05);石油醚粗提物的抑制作用最小,均在10%以下.小桐子果壳和枝叶水粗提物的石油醚、氯仿、乙酸乙酯、正丁醇和水萃取物(10 g·L~(-1))对萝卜和苏丹草幼苗的根长和茎高均有不同程度的抑制作用,其中水、正丁醇和乙酸乙酯萃取物的抑制作用显著高于氯仿和石油醚萃取物,抑制率均在70%以上;水萃取物的抑制作用最强,抑制率均在80%以上;石油醚萃取物的抑制作用最小,抑制率均在10%以下.研究结果表明,小桐子果壳和枝叶的水粗提物具有一定的除草活性,其有效成分为极性较大的组分.     

亚稀褶黑菇对小白鼠肝肾超微结构的影响

对小白鼠用亚稀褶黑菇提取液进行毒性试验．测定了亚稀褶黑菇的半致死剂量LD50值;应用透射电镜观察了小白鼠亚稀褶黑菇中毒后的病理变化,结果表明．亚稀褶黑菇的LD50值为2852．1mg／kg,电镜检查发现。亚稀褶黑菇毒素主要损害肾脏,引起肾细胞核核膜肿胀．线粒体肿胀,溶酶体增多,肾细胞坏死．     

Production of 2-(2-phenylethyl) Chromones in Cell Suspension Cultures of <Emphasis Type="Italic">Aquilaria sinensis</Emphasis>

2-(2-Phenylethyl) chromones are the major constituents responsible for the quality of agarwood, which is one of the most valuable non-timber products used as incenses, perfumes, traditional medicines and other products. In this study, cell suspension culture of Aquilaria sinensis (Lour) Gilg was used to monitor the eliciting effects of crude fungal extracts on cell growth and chromones production. Crude extracts of Melanotus flavolivens (B. etc) Sing. prepared with different solvents were used to elicit the production of 2-(2-phenylethyl) chromones in cell suspension cultures of A. sinensis. Four 2-(2-phenylethyl) chromones,␣6,7-dimethoxy-2-(2-phenylethyl) chromone (1), 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl] chromone (2), 6-methoxy-2-[2-(4′-methoxyphenyl)ethyl] chromone (3) and 6-methoxy-2-(2-phenylethyl) chromone␣(4),␣were detected by LC–MS in the cell suspension culture of A. sinensis elicited with crude extracts of M. flavolivens. Three hundred and seventy eight, 196 and 31 μg g⁻¹ DW of 2-(2-phenylethyl) chromones were obtained in the cell cultures induced by water extracts, 50 and 95% ethanol extracts of M. flavolivens, respectively. The results show that water-soluble materials in the crude extracts are the main components inducing the production of 2-(2-phenylethyl) chromones in the cell cultures.     

Inhibition of Oxidative Stress and Lipid Peroxidation by Anthocyanins from Defatted Canarium odontophyllum Pericarp and Peel Using In Vitro Bioassays

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD⁺ and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection.     

Malate: A Possible Source of Error in the NAD Glutamate Dehydrogenase Assay

The effects of externally induced metabolic perturbations areoften studied through changes of the enzyme activity patternsin crude plant extracts. From glutamate dehydrogenase (GDH)it is reported that environmental changes not only influencethe amount of the enzymatic activity, but also the ratio ofthe aminating to the deaminating activities (NADH/NAD⁺ ratio).Using crude cell extracts of suspension cultures of wheat (Triticumaestivum L. cv. Heines Koga II) we find evidence that the pretreatmentof the homogenate directly influences this ratio. Dialysis ofthese crude cell extracts resulted in a 70% loss of the NAD⁺activity, while the NADH activity remained unchanged. The deaminatingactivity in the dialysed extract could be completely restoredupon addition of a dialysable factor which was identified tobe malate. The interference of malate with the glutamate dehydrogenasereaction is caused through the action of malate dehydrogenaseand glutamate oxaloacetate transaminase which are both presentin high activities in the extracts. Only in exhaustively dialysedcell extracts can the proper deaminating GDH activity be determined.The results are discussed in the light of the controversialreports on the variable ratio of the NADH/NAD⁺ activity of GDH. Key words: Glutamate dehydrogenase, malate, NADH/NAD⁺, activity, Triticum aestivum     

Enzymatic Removal of Diacetyl from Beer: II. Further Studies on the Use of Diacetyl Reductase1

Diacetyl removal from beer was studied with whole cells and crude enzyme extracts of yeasts and bacteria. Cells of Streptococcus diacetilactis 18-16 destroyed diacetyl in solutions at a rate almost equal to that achieved by the addition of whole yeast cells. Yeast cells impregnated in a diatomaceous earth filter bed removed all diacetyl from solutions percolated through the bed. Undialyzed crude enzyme extracts from yeast cells removed diacetyl very slowly from beer at its normal pH (4.1); at a pH of 5.0 or higher, rapid diacetyl removal was achieved. Dialyzed crude enzyme extracts from yeast cells were found to destroy diacetyl in a manner quite similar to that of diacetyl reductase from Aerobacter aerogenes, and both the bacterial and the yeast extracts were stimulated significantly by the addition of reduced nicotinamide adenine dinucleotide (NADH). Diacetyl reductase activity of four strains of A. aerogenes was compared; three of the strains produced enzyme with approximately twice the specific activity of the other strain (8724). Gel electrophoresis results indicated that at least three different NADH-oxidizing enzymes were present in crude extracts of diacetyl reductase. Sephadex-gel chromotography separated NADH oxidase from diacetyl reductase. It was also noted that ethyl alcohol concentrations approximately equivalent to those found in beer were quite inhibitory to diacetyl reductase.     

Epitope masking of rat esophageal carcinoma tumor-associated antigen by certain coexisting glycolipid and phospholipid molecules: a potential mechanism for tumor cell escape from the host immune responses

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.     

Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX(®) test

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.     

A procedure to remove protease activities from Bacillus subtilis sporulating cells and their crude extracts.

A simple procedure was developed to remove both extracellular and intracellular proteases associated with aporulating Bacillus subtilis cells. Cells are washed four times with 1 m KCl before breakage and their crude extracts are treated with 2 mm diisopropylfluorophosphate before passage through a hemoglobin-Sepharose affinity column. RNA polymerase in crude B. subtilis extracts treated by this procedure was stable, functionally and structurally, for more than 1 month at 4°C. This process for removing all proteases should work essentially with any crude extracts containing proteolytic activities.