Isolation of a New Cytokinin from Immature Yellow Lupin Seeds

A new cytokinin was isolated from methanol extracts of immature lupin seeds (Lupinus luteus) through successive purification of charcoal adsorptions, silver precipitations, ion exchange and paper chromatography. The structure was confirmed by spectral data and synthesis as (?)-6-N-(4-hydroxy-3-methylbutylamino)purine, (?)-dihydrozeatin (I).     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

Conversion of (−)-Carvotanacetone and (+)-Carvotanacetone by Pseudomonas ovalis,Strain 6–1

As a part of series on the biochemical reduction of terpenes, the conversion of (?)-carvotanacetone (I) and (+)-carvotanacetone (II) by Pseudomonas ovalis, strain 6–1, has been studied.

By the action of the microorganism, I was reduced to give (+)-carvomenthone (III), (+)-neocarvomenthol (IV), and (?)-carvomenthol (V), whereas II was also reduced to (?)-isocarvomenthone (VI), (?)-carvomenthone (VII), (?)-isocarvomenthol (VIII), and (?)-neoisocarvomenthol (IX); of which III, VI and IX are the major products.

The metabolic pathways of I and II and mechanism of stereospecific hydrogenation are also discussed.     

Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.     

Isopenicillin N epimerase activity in a high cephalosporin-producing strain of Cephalosporium acremonium

Summary Isopenicillin N epimerase activity in Cephalosporium acremonium CW-19 is so labile that it has never been detected in sonic extracts. Prior to this work, it had only been obtained by the laborious protoplast lysate procedure. The present work shows that the enzyme is present in sonic extracts of a high cephalosporin-producing strain (C-10) of C. acremonium throughout the fermentation.     

Effects of extracts from sporoderm-broken spores of Ganoderma lucidum on HeLa cells

The effects of extracts from Ganoderma lucidum spores on the growth of human cervix uteri tumor HeLa cells as well as on the cell cycle and intracellular calcium level were investigated. Alcohol extracts were prepared from sporoderm-broken and sporoderm-nonbroken spores (termed extract I and extract II) of G. lucidum. Extract I was then subjected to silica gel chromatography to obtain extract III. Cytotoxicity was examined by means of trypan blue exclusion and MTT tests. It was found that extract I and extract III, but not extract II strongly inhibited the growth of HeLa cells, and that extract III was more effective than extract I. Moreover, extract III was shown to be capable of blocking the cell cycle at the transition from G₁ to S phase and inducing a marked decrease of intracellular calcium level, determined by flow cytometry and the specific fluorescent calcium probe Fura-2, respectively. These results imply that (1) the breaking of G. lucidum spores improves the release of cytotoxic activity and (2) the effective extract might influence the cell cycle and cellular signal transduction by altering the calcium transport system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pluripotency of a polyploid H1 (ES) cell system without leukaemia inhibitory factor

Objectives: Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)‐cell system was prepared. Materials and methods: Diploid, tetraploid, pentaploid, hexaploid, octaploid and decaploid H1 (ES) cells (2H1, 4H1, 5H1, 6H1, 8H1 and 10H1 cells, respectively) were cultured for about 460 days in L15F10 medium without leukaemia inhibitory factor (LIF). The cells cultured under LIF‐free conditions were denoted as 2H1(?), 4H1(?), 5H1(?), 6H1(?), 8H1(?) and 10H1(?) cells, respectively. Pluripotency and gene expression were examined. Results: Ploidy alteration of H1(?) cells was similar to that of H1 cells. The polyploid H1(?) cells showed positive activity of alkaline phosphatase, suggesting that they maintained pluripotency in vitro without LIF. The polyploid H1(?) cells formed teratocarcinomas in mouse abdomen, suggesting they could differentiate in mouse abdomen in vivo. 2H1, 4H1 and polyploid H1(?) cells expressed nanog, oct3/4 and sox2 genes, suggesting that they fulfilled the criteria of ES cells. Nanog gene was significantly over‐expressed in 4H1 and polyploid H1(?) cells, suggesting that overexpression of nanog gene was a characteristic of polyploid H1 cells. Conclusion: Polyploid H1 (ES) cells retained pluripotency in vitro, without LIF with nanog over‐expression.     

Aggregation pheromone of the Oriental spruce engraver Pseudips orientalis

• 1 Volatiles from the hindgut extracts of males of the Oriental spruce engraver Pseudips orientalis (Wood & Yin) (Coleoptera: Curculionidae, Scolytinae) of different phases of gallery development were analyzed by gas chromatography‐mass spectrometry‐flame ionization detection (GC‐MS/FID) with both polar and enantioselective columns.
• 2 GC‐MS/FID analyses showed that unmated males or males mated with one female produced approximately 95%‐(?)‐ipsenol and (?)‐cis‐verbenol as major components, as well as (?)‐trans‐verbenol, myrtenol, approximately 70%‐(+)‐ipsdienol and (?)‐verbenone as minor or trace components. The release of these male‐produced compounds was confirmed by GC analysis of an aeration sample of a P. orientalis‐infested spruce log. Mating reduced production of the male‐specific hindgut volatiles.
• 3 A field‐trapping bioassay in Qinghai, China, showed that a ternary blend containing two major components, 97%‐(?)‐ipsenol (i.e. close to naturally produced enantiomeric ratio) and (?)‐cis‐verbenol, plus a minor component (?)‐trans‐verbenol, caught significantly more P. orientalis beetles (♂: ♀ = 1: 2.7) compared with the unbaited control. Subtraction of (?)‐trans‐verbenol from the active ternary blend had no significant effect on trap catches. The addition of (±)‐ipsdienol (at 0.2 mg/day release) to the active ternary or binary blends significantly interrupted their trap catches. Replacing 97%‐(?)‐ipsenol with (±)‐ipsenol in the ternary blend significantly reduced trap catches to a level that was no different from the blank control.
• 4 Pseudips species were sister to all other Ipini genera in a phylogeny reconstructed with mitochondrial cytochrome oxidase I DNA data for 51 Ipini and outgroup species.
• 5 The results obtained suggest that the two major components, 95%‐(?)‐ipsenol and (?)‐cis‐verbenol (at approximately 4–5 : 1), produced by unmated fed males, are probably the primary aggregation pheromone components for P. orientalis. In light of the phylogeny, the use of terpenoid semiochemicals as pheromones probably occurred early in the evolution of Ipini and these semiochemical blends were subsequently modified in the process of speciation.

     

Caffeoylquinic Acids,Cytotoxic, Antioxidant,Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria

Chlorogenic (5‐CQA), 1,5‐, 3,5‐, 4,5‐ and 3,4‐dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus‐christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess .) DC. and I. germanica L. by HPLC analysis. The amount of 5‐CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5‐DCQA (4.05–55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5‐DCQA, 4,5‐DCQA and 3,4‐DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS^.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 μg/mL toward non‐cancer (MDCK II) and cancer (A 549) cells.     

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7′-epoxylignan and the introduction of apoptosis by caspase 3/7

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (?)-verrucosin, which is (7S,7′R,8R,8′R)-7,7′-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC₅₀ = 6.6 μM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (?)-verrucosin 13, the 7-(4-methoxyphenyl)-7′-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC₅₀ = 2.4 μM) is three times greater than (?)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (?)-verrucosin derivative.     

A novel polygalacturonic acid bioflocculant REA-11 produced by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: a proposed biosynthetic pathway and experimental confirmation

Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 M UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R²=0.97), UDP-galactose epimerase (R²=0.75) and UDP-galactose dehydrogenase (R²=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.     

Pyruvate Decarboxylase Catalysed Formation of Terpenoid α-hydroxy Ketones

Enzyme extracts of the wild type yeast Zygosaccharomyces bisporus were applied for the pyruvate decarboxylase catalysed condensation of pyruvate and (R)-(+)-and (S)-(?)-perillyl aldehyde, (±)-citronellal, neral, geranial or (R)-(?)-myrtenal to form novel α-hydroxy ketones. Best yields were obtained when the transformation medium contained 25% (v/v) of the cosolvent N,N-dimethylformamide. Conversion of (R)-(+)-perillyl aldehyde to (1R)-1-hydroxy-1-[(4’R)-4’-isopropenyl-1-cyclohexen-1-yl]-2-propanone proceeded highly stereospecifically (>99% de), whereas the stereoselectivity was somewhat less in the transformation of (S)-(?)-perillyl aldehyde (58% de) and (R)-(?)-myrtenal (92% de). All of the new compounds imparted characteristic odour impressions as determined by means of GC-olfactometry.     

Two new cell lines originated from the embryos of <Emphasis Type="Italic">Clostera anachoreta</Emphasis> (Lepidoptera: Notodontidae): characterization and susceptibility to baculoviruses

Two cell lines designated CAF-Clan I and CAF-Clan II have been established from embryos of Clostera anachoreta (Lepidoptera: Notodontidae) in TNM-FH medium containing 10% inactivated fetal bovine serum. CAF-Clan I consists of a mixture of three cell types: spherical cells, spindle-shaped cells, and giant cells. Most of the cultured cells formed a suspension in the medium and were subcultured more than 60 passages. CAF-Clan II mainly consists of spindle-shaped and spherical cells which attached to the culture surface and have undergone more than 40 passages. The cell population doubling time at 27°C of CAF-Clan I at passage 22 and CAF-Clan II at passage 24 was about 68.5 and 38.2 h, respectively. The chromosome number of both cell lines at passage 15 varied from 62 to 100 in the majority of cells, though a few cells exceeded 260 (n = 30). DNA amplification fingerprinting–polymerase chain reaction analysis confirmed that the origination of the two cell lines was C. anachoreta. The susceptibility of the cell lines to baculoviruses was tested. The results showed that CAF-Clan II was susceptible to infection of Autographa californica nucleopolyhedrovirus (AcMNPV) and Ecotropis oblique nucleopolyhedrovirus (EoNPV). Occlusion bodies (OBs) production was 129 ± 4 OBs/cell and 124 ± 15 OBs/cell for AcMNPV and EoNPV, respectively. CAF-Clan I was less susceptible to AcMNPV compared with CAF-Clan II, while non-permissive to EoNPV.     

Population divergence of aggregation pheromone responses in Ips subelongatus in northeastern China

The Asian larch bark beetle, Ips subelongatus, is considered to be the major pest of larch within its natural range. We investigated the electrophysiological and behavioral characteristics as well as mitochondrial DNA cytochrome oxidase subunit I sequences of I. subelongatus from 13 geographic populations throughout northeastern China in order to explore population divergence of aggregation pheromone responses and the extent of potential genetic divergence. Electrophysiological analyses showed that antennae of I. subelongatus from all the six tested populations responded strongly to (S)‐(?)‐ipsenol (100% detection; 0.35–0.73 mV) in gas chromatography (GC)–electroantennographic detection (EAD) analyses, while its antipode, (R)‐(+)‐ipsenol was antennally inactive. I. subelongatus populations varied in their responses to (R)‐(?)‐ and (S)‐(+)‐ipsdienol in GC‐EAD analyses. Behavioral bioassays demonstrated that (S)‐(?)‐ipsenol alone was significantly attractive at all the tested sites, supporting its status as a key pheromone component of I. subelongatus, whereas (S)‐(+)‐ipsdienol was inactive alone. Adding (S)‐(+)‐ipsdienol to (S)‐(?)‐ipsenol did not have any effect on the trap catches from some populations in Inner Mongolia. However, (S)‐(+)‐ipsdienol showed a strong synergistic effect on (S)‐(?)‐ipsenol from several populations in Jilin and Liaoning Provinces, and a weak synergistic effect from some transition populations in Heilongjiang Province. Furthermore, 27 mitochondrial haplotypes were found among the 13 populations (intraspecific nucleotide divergence, 0.1%–1.1%). Analyses of molecular variance and haplotype networks indicated that different geographic populations have developed some genetic variation but did not form completely independent groups. From an applied point of view, a universal synthetic binary blend of racemic ipsenol and (S)‐(+)‐ipsdienol might have a potential for monitoring or even mass‐trapping of I. subelongatus across northeastern China, even though some populations only use (S)‐(?)‐ipsenol alone as their active pheromone component.     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.     

Stereoselective protein binding of tetrahydropalmatine enantiomers in human plasma,HSA, and AGP,but not in rat plasma

Tetrahydropalmatine (THP) is one of the active alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC‐UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)‐THP was significantly higher than that of (?)‐THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)‐THP, expressed as nK_A, were 9.0 × 10³ M^?1 and 2.34 × 10⁵ M^?1, respectively, which were notablely higher than to (?)‐THP, with the nK_A of 3.4 × 10³ M^?1 and 1.44 × 10⁵ M^?1, respectively. The binding site of HSA for (?)‐THP was Site I, whereas for (+)‐THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (?)‐ and (+)‐THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (?)‐ and (+)‐THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (?)‐ and (+)‐THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug–drug interaction on THP in healthy human plasma. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Field response of the northern spruce bark beetle Ips duplicatus (Sahlberg) (Coleoptera: Curculionidae,Scolytinae) to different combinations of synthetic pheromone with (−)‐α‐pinene and (+)‐limonene

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Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.