Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues β2-adrenoceptor using an affinity chromatographic selection method

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β₂-adrenoceptor (β₂-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β₂-AR affinity column. The retention compounds eluted from the β₂-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β₂-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β₂-AR. Their association constants were (2.28 ± 0.11) × 10⁴/M and (3.00 ± 0.10) × 10⁴/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β₂-AR, and the corresponding association constants were (2.20 ± 0.09) × 10⁻⁴/M and (6.78 ± 0.001) × 10⁵/M.     

Characterization and antioxidant activity of Ginkgo biloba exocarp polysaccharides

Ginkgo biloba exocarp polysaccharide (GBEP) was obtained by hot water extraction, the crude polysaccharide was deproteinized by Sevag method and fractionized by a DEAE Sepharose fast flow anion-exchange column. Five fragments were obtained, including neutral polysaccharide (GBEP-N) and four acidic polysaccharides (GBEP-A1, GBEP-A2, GBEP-A3 and GBEP-A4). GBEP-N and GBEP-A3 were further purified by Superdex 200 gel column chromatography. The resulted two fractions GBEP-NN, and GBEP-AA were characterized by FT-IR, and HPGFC (high pressure gel filtration chromatography). Monosaccharide composition was determined by RP-HPLC method of precolumn derivatization with 1-phenyl-3-5-pyrazolone. GBEP-NN was mainly composed of rhamnose, arabinose, mannose, glucose and galactose, while GBEP-AA was mainly made up of mannose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose. The crude GBEP exhibited certain antioxidant activity. At the concentration of 5 mg/mL, the hydroxyl radical scavenging effect of GBEP was 90.52%, greater than 77.37% for the positive control ascorbic acid.     

Community structure and food web based on stable isotopes (δN and δC) analysis of a North Eastern Atlantic maerl bed

Maerl beds are highly biodiverse biogenic substrata that have been receiving increasing attention in the last decade. Although maerl beds represent important nursery areas for commercial fishes and molluscs, little is known on the trophic web of their communities. Community structure parameters of maerl bed of the Bay of Brest (species richness, abundance, biomass and dominating species) were studied in parallel with the carbon and nitrogen isotopic composition of their main benthic species (macrofaunal, and megafaunal organisms) in order to assess the trophic levels and differences in the potential food sources of maerl inhabitants. The major potential sources of energy were identified to originate either from epiphytic macroalgae and microphytobenthos both growing on maerl thalli, together with sedimenting (sedimentary) particulate organic matter (POM) originating from the water column. The majority of the macro- and megafaunal organisms investigated were filter feeders, selective-deposit feeders and predators/scavengers. Filter feeders fall into three different groups representing different trophic pathways (i) sponges feeding directly on POM (water column filter feeders I), (ii) ascidians and holothurians feeding on POM and probably captured pelagic preys (water column filter feeders II), and (iii) filter feeding molluscs and crustaceans were hypothesised to feed on microphytobenthos or on decaying sedimented POM (Interface filter feeders). Selective deposit feeders were also divided into two subgroups. Carnivores were also distinguished between those with scavenging habits and true predators. Coupling of the trophic levels observed with the community biomass structure revealed that most of the benthic biomass derives its food from detritic sedimented POM and/or microphytobenthos, with interface filter feeders (23% of the biomass), selective deposit feeders (12%). Carnivores made up to 14% of the total biomass. Generally stable isotopes ratio mean values overlap and cover a large range within feeding types, indicating a strong overlap in food sources and a high degree of complexity of the food web presumably due to the diversity of the potential food sources.     

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.     

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.     

Polyhydroxylated steroids from the soft coral Sinularia dissecta

A repeated silica gel column chromatography followed by HPLC purification on the methanol extract of marine soft coral Sinularia dissecta, resulted in the isolation of fifteen polyhydroxylated steroids (1-15), involving six new C-18 functionalized steroids, 3beta-acetoxy-1alpha,11alpha-dihydroxygorgost-5-en-18-oic acid (1), gorgost-5-en-1alpha,3beta,11alpha,18-tetrol (2), 18-acetoxy-1alpha,3beta,11alpha-trihydroxygorgost-5-ene (3), 24(S)-3beta-acetoxy-1alpha, 11alpha-dihydroxyergost-5-en-18-oic acid (4), 24(S)-ergost-5-en-1alpha,3beta,11alpha,18-tetrol (5), and dissectolide (6). The structures of the new compounds were determined on the basis of extensive spectroscopic data (IR, MS, (1)H and (13)C NMR, HMQC, HMBC, and NOESY) analysis. Compound 6 was found as an unusual sterol bearing a lactone functionality.     

Two glucosylated abscisic acid derivates from avocado seeds (Persea americana Mill. Lauraceae cv. Hass)

Phytochemical investigation of avocado seed material (Persea americana Mill., Lauraceae) resulted in the isolation of two glucosylated abscisic acid derivates. One of these was not known as a natural product and can be regarded as a potential 'missing link' in abscisic acid metabolism in plants. After fractionation by high-speed countercurrent chromatography, and multiple steps of column chromatography, structures were elucidated by 1D-, 2D-NMR, electrospray-MS to be the novel beta-d-glucoside of (1'S,6'R)-8'-hydroxyabscisic acid, and (1'R,3'R,5'R,8'S)-epi-dihydrophaseic acid beta-d-glucoside. Absolute configuration was determined by circulardichroism, optical rotation, and by NOE experiments.     

Separation of floridoside and isofloridosides by HPLC and complete (1)H and (13)C NMR spectral assignments for D-isofloridoside

Isofloridosides (1-O-alpha-D-galactopyranosylglycerol) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga Porphyra umbilicalis (Linné) Kützing (Bangiales, Rhodophyta). Their separation was achieved by HPLC (NH(2) P50 column) after successive purification of the crude extract by ion-exchange chromatography and HPLC (Sugar-Pak TM1 column). 1D and 2D NMR spectroscopy experiments allowed to completely assign the (1)H and (13)C spectra of D-isofloridoside.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.     

Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase

Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.     

山溪鲵的脊柱特征,兼议有尾目脊柱的划分

为探讨有尾目脊椎的划分,本文以小鲵科的山溪鲵(Batrachuperus pinchonii)为例,运用透明骨骼双色法对其脊柱的形态特征进行了观察,并对各部分椎骨特征进行详细描述和绘图.结果显示,山溪鲵的脊椎根据椎骨是否具前关节突、横突、肋骨、肋软骨和脉弓等形态特征可分5部分;同时结合小鲵科其他20种94号标本和蝾螈科6种27号标本的脊柱特征及文献资料,讨论了有尾目脊椎的划分,认为将有尾目脊柱划分为5部分(颈椎、躯椎、荐椎、尾荐椎和尾椎)的观点较将其划分为4部分(颈椎、躯椎、荐椎和尾椎)的观点更合理.     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.     

p-Terphenyls from the fruiting bodies of Paxillus curtisii and their antioxidant properties

New p-terphenyls, curtisians R (1)–V (5), have been isolated together with known compounds, curtisians E, I–P, and kynapcin-12, from the methanolic extract of the fruiting bodies of Paxillus curtisii (Paxillaceace). Their structures were established by various spectroscopic analyses including 1D- and 2D-NMR experiments, as well as high-resolution FAB-mass analysis. They exhibited significant protective effects against oxidative damage of supercoiled DNA and 2-deoxyribose by hydroxyl radicals generated from the Fenton reaction.     

The absolute configuration of (-)-3-hydroxy-alpha-calacorene

3-Hydroxy-alpha-calacorene was identified in extracts from cold-shocked seedlings of cotton (Gossypium hirsutum L.) and kenaf (Hibiscus cannabinus L.), both of which are members of the Malvaceae family. (-)-3-Hydroxy-alpha-calacorene was isolated from Heterotheca inuloides Cass. (Asteraceae). HPLC on a chiral stationary phase column showed that the 3-hydroxy-alpha- calacorene from cotton and kenaf had the same relative configuration, while that from H. inuloides was of the opposite configuration. X-ray crystallographic analysis established the absolute configuration of the compound in H. inuloides as (8R)-(-)-3-hydroxy-alpha-calacorene.     

Environmental factors regulating the radial oxygen loss from roots of Myriophyllum spicatum and Potamogeton crispus

Myriophyllum spicatum and Potamogeton crispus are common species of shallow eutrophic lakes in north-eastern Germany, where a slow recovery of the submersed aquatic vegetation was observed. Thus, the characterisation of the root oxygen release (ROL) as well as its implication for geochemical processes in the sediment are of particular interest. A combination of microelectrode measurements, methylene blue agar and a titanium(III) redox buffer was used to investigate the influence of the oxygen content in the water column on ROL, diel ROL dynamics as well as the impact of sediment milieu. Oxygen gradients around the roots revealed a maximum oxygen diffusion zone of up to 250 μm. During a sequence with a light/dark cycle as well as alternating aeration of the water column, maximum ROL with up to 35% oxygen saturation at the root surface occurred under light/O₂-saturated conditions. A decrease to about 30% was observed under dark/O₂-saturated conditions, no ROL was detected at dark/O₂-depleted conditions and only a weak ROL with 5–10% oxygen saturation at the root surface was measured under light but O₂-depleted water column. These results indicate, that during darkness, ROL is supplied by oxygen from the water column and even during illumination and active photosynthesis production, ROL is modified by the oxygen content in the water column. Visualisation of ROL patterns revealed an enhanced ROL for plants which were grown in sulfidic littoral sediment in comparison to plants grown in pure quartz sand. For both plant species grown in sulfidic littoral sediment, a ROL rate of 3–4 μmol O₂ h⁻¹ plant⁻¹ was determined with the Ti(III) redox buffer. For plants grown in pure quartz sand, the ROL rate decreased to 1–2 μmol O₂ h⁻¹ plant⁻¹. Hence, aside from the oxygen content in the water column, the redox conditions and microbial oxygen demand in the sediment has to be considered as a further major determinant of ROL.     

Implications of a fluctuating fish predator guild on behavior, distribution, and abundance of a shared prey species: the grass shrimp Palaemonetes pugio

In some systems, the identity of a prey species' dominant predator(s) may not be constant over time. In cases in which a prey species exhibits different responses to various predator species, such changes in predator identity may have population-wide consequences. Our goals were to determine (1) whether mortality of and refuge use by the grass shrimp, Palaemonetes pugio, were predator-specific, and (2) how effects of prey size and habitat interacted with predator type. Striped bass (Morone saxatilis) exerted twice as much predation pressure as mummichog (Fundulus heteroclitus), although not equally as great on large (female) and small (male) shrimp. Mummichog, which fed preferentially on large shrimp, forced a partitioning of habitat between the two shrimp size classes. In contrast, large and small shrimp occupied similar habitats when subjected to striped bass, which fed on both size classes equally. Refuge use of grass shrimp depended on predator type. In the presence of mummichog, which occupied shallower depths in the water column than striped bass, shrimp stayed deep and close to structural habitat. Striped bass, which were deeper, caused shrimp to move high in the water column away from structural habitat. When both predators were present, shrimp distribution was similar to that when only striped bass were present, striped bass predation rate was enhanced, and overall mortality was higher than with either predator alone. Results suggest that at times when mummichogs are the dominant predators, large (female) shrimp experience higher predation than small (male) shrimp and are physically separated from their potential mates. When striped bass are more abundant, male and female shrimp may share a similar, shallow, less structure-oriented distribution and be subjected to higher mortality. When both predators are present, mortality rates may be higher still. This predator-, size-, and habitat-specificity of grass shrimp behavior suggests significant population and distribution consequences of fluctuating predator guilds and fluctuating cover of structural habitats in the field.     

Significance of oxygen supply in production of a novel antibiotic by Pseudomonas sp. SJT25

A new agricultural antibiotic, 2-heptyl-5-hexylfuran-3-carboxylic acid (HHCA), was recently discovered, but its further application was limited due to its low production titer. In shake flask fermentation, the effect of initial K(L)a within the range of 2.12-18.87 h?1 on HHCA production was investigated. The cell growth and glycerol consumption were faster at a higher initial K(L)a value, but the maximum production (14.43 mg/L) was attained at an initial K(L)a value of 12.46 h?1. The oxygen supply information was further applied to a 2-L bubble column bioreactor (BCB) by varying initial K(L)a from 1.45 to 30.18 h?1, and the hyperproduction of HHCA was achieved at a relatively low initial K(L)a around 5-10 h?1. The control of oxygen supply is considered to be an important strategy to enhance HHCA production, and the information obtained will be useful to production of this powerful new antibiotic on a large scale.     

白木香果实化学成分研究

运用柱色谱技术从白木香(Aquilaria sinensis (Lour.) Gilg)果实中分离得到7个化合物,经波谱解析和理化性质分别鉴定为: 3-吲哚甲酸(1)、6-羟基-2-[2-(4-羟基苯基)乙基]色原酮(2)、芫花素(3)、4', 5-二羟基-3',7-二甲氧基黄酮(4)、洋芹素-7,4'-二甲醚(5)、 β-谷甾醇(6)和胡萝卜苷(7)。其中化合物1为首次从瑞香科沉香属植物中分离得到。     

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.     

Structural analysis of a unique hybrid-type ganglioside with isoglobo-, neolacto-, and ganglio-core from the gills of the Pacific salmon (Oncorhynchus keta)

Monosialosyl gangliosides from the gills of the Pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. The unknown acidic glycolipids (M14 and M15) with slower mobility than GM1a on thin-layer chromatography were separated by Iatrobeads column chromatography and were characterized by compositional analysis, methylation analysis, chemical, and enzymatic degradation, negative-ion LSIMS, and (1)H nuclear magnetic resonance spectroscopy. Both M14 and M15 contained a same oligosaccharide core with isoglobo-, neolacto-, and ganglio-series as follows: [carbohydrate structure: see text]. The only difference between M14 and M15 was in fatty acid acylation. Analysis of the fatty acids indicated a predominance of C24:1 fatty acid in M14 and shorter chain saturated fatty acids, C14:0 and C16:0, in M15.