Influence of phenolic compounds on Agrobacterium vir gene induction and onion gene transfer

Eight chalcones and benzalacetones were tested for their virulence induction on Agrobacterium tumefaciens. With one exception, they had a strong action, and in particular 4-(3,5-dimethoxy-4-hydroxy-phenyl)-but-3-en-2-one (10a) was very effective with a virulence induction about 1.5–2 times that of acetosyringone (AS). When applied to Agrobacterium-mediated gene transfer of onion, both 10a and AS at 250 μM, led to increased gene transfer of between 25 and 35% when compared with the control. Many derivatives of AS were tested and the indispensable chemical functions required to induce vir genes were determined; for example, the presence of at least one methoxy group and a carbonyl group as in acetyl, aldehyde and acid functions. The most effective vir-inducing compound used was the original AS with two methoxy groups and an acetyl function. By testing the corresponding β-glucosides and glucosyl esters (from acids), we established that a phenolic function was also essential for virulence induction. These glucosides led to a decreased toxicity to the bacteria in relation to the original product. However, the presence of a β-glucosylated phenol function led to the total loss of vir induction while the corresponding esters, particularly the glucosyl sysingate ester, conserved a good vir induction.     

Directional Modifications of Resibufogenin by Mucor subtilissimus and Pseudomonas aeruginosa

Directional modifications of resibufogenin 1 by Mucor subtilissimus and Pseudomonas aeruginosa were carried out. The substrate was hydroxylated at C-12 by M. subtilissimus AS 3.2454, from which a major product 12-hydroxyresibufogenin 2 was obtained. Then product 2 was dehydrogenated by P. aeruginosa AS 1.860, which resulted in a new compound 12β-hydroxy-3-keto-resibufogenin 3.     

虾青素复合有氧运动对D-半乳糖诱导大鼠肾脏衰老的干预作用及其机制

目的:研究虾青素复合有氧运动对D-半乳糖诱导大鼠肾脏衰老的干预作用及其机制.方法:60只3月龄SPF级SD大鼠采用两因素两水平2×2析因设计随机分为空白对照组(C组)、急性衰老组(S组)、虾青素+急性衰老组(AS组)、有氧运动+急性衰老组(ES组)、虾青素+有氧运动+急性衰老组(AES组),每组12只.大鼠腹腔注射10...     

Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31–40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30–39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-γ production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.     

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30–50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab′)₂ fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl). A peak containing primarily the F(ab′)₂ antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.     

The Production of Catechols from Benzene and Toluene by Pseudomonas Putida in Glucose Fed-Batch Culture

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1^-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1^-1).

In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibited within 4-6 hours of the maximum product concentration being attained.

3-Methylcatechol overproduction from toluene was also studied using a continuous product removal system. Granular activated charcoal removed 3-methylcatechol efficiently and was easily regenerated by washing with ethyl acetate. Using P. putida 2313, it was shown that the final product concentration increased approximately fourfold. Additional products were formed and the significance of these are discussed.     

Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 μg/ml for anti-diabetic activity in peripheral tissues and pancreatic β cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in β TC-tet pancreatic β cells; (3) β cell proliferation in β TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15–25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested β TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase ³H-thymidine incorporation in replicating β TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20–33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in β cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.     

Effect of the homokaryotic or heterokaryotic state of the uvs-2 allele in Neurospora crassa on mitomycin C-induced killing and ad-3 mutation

Mitomycin C (MC) was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 4 dikaryons of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on MC-induced killing and ad-3 mutation. These dikaryons were homokaryotic for uvs-2⁺ (H-12), homokaryotic for uvs-2 (H-59), and heterokaryotic for uvs-2/uvs-2⁺ (H-70 and H-71). MC induced killing and ad-3 mutation in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a great increase in the killing and mutagenic activities of MC. This increased sensitivity to MC-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is a different effect than that noted by others for a defect in nucleotide excision-repair in Escherichia coli and Salmonella typhimurium or in human cells. The dikaryons heterokaryotic for uvs-2/uvs-2⁺ had the same sensitivity to MC as H-12, indicating that for MC-induced killing and ad-3 mutation uvs-2 is recessive to uvs-2⁺.     

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bishydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.     

决明GRAS基因家族全基因组鉴定及其在盐和干旱胁迫条件下的表达分析

目的: 基于决明(Senna tora L.)全基因组数据,对GRAS家族成员、理化性质、基因结构、进化关系以及胁迫条件下的表达模式进行鉴定和分析。方法: 将决明基因组蛋白数据与拟南芥GRAS成员进行比对,分别利用TBtools、MEGA-X、CLUSTALW、MEME等生物信息学软件和工具,对决明GRAS基因家族成员进行分析。利用qRT-PCR(quantitative real-time PCR)检测干旱和盐胁迫条件下决明根中GRAS基因的表达情况。结果: 50个StGRAS分为9个亚家族,不均等地分布在13条染色体上。结构分析表明,StGRAS34和StGRAS12分别与蒺藜苜蓿(Medicago truncatula)结瘤信号蛋白NSP1和NSP2高度同源。StGRAS的启动子区域多含有与胁迫响应、激素调节等相关的响应元件。qRT-PCR结果表明,在盐胁迫条件下,StGRAS表达具有明显差异;在干旱胁迫条件下,绝大多数检测基因能够快速响应,表达显著升高;两种胁迫条件下,StGRAS28和StGRAS29表达趋势互补,具有协同调控关系。结论: GRAS基因家族能够广泛参与胁迫响应,其中StGRAS28与StGRAS29可能共同参与介导决明根的盐与干旱胁迫应答,StGRAS34和StGRAS12分别作为决明共生结瘤的NSP1和NSP2,可能与增强结瘤因子信号诱导相关,这为进一步挖掘和研究GRAS基因在决明响应胁迫和共生固氮过程所发挥的作用提供了基础。     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Oligosaccharide and alkyl β-galactopyranoside synthesis from lactose with Caldocellum saccharolyticum β-glycosidase

The synthetic utility of the thermostable β-glycosidase from Caldocellum saccharolyticum was investigated. The ability of the enzyme to catalyze oligosaccharide and β-galactopyranoside synthesis from lactose was compared with that of the readily commercially available, moderately thermostable β-galactosidase (β- -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae. Generally, the C. saccharolyticum enzyme showed significantly greater resistance to inactivation by heat and organic solvent and better yields of product. Although the A. oryzae enzyme gave better oligosaccharide yields at lower lactose concentrations, at higher concentrations (above 50% w/w) the C. saccharolyticum enzyme was significantly better, yielding a sugar mixture containing 42% by weight of tri- plus tetra-saccharides, from a 70% w/w lactose solution, compared with 31% by weight of oligosaccharides with the A. oryzae enzyme. In ethyl galactoside synthesis from ethanol and lactose, neither enzyme appeared to hydrolyze the product to any great extent. Under all conditions tested, the product yield did not peak, even at long reaction times, when most of the lactose had been consumed. The C. saccharolyticum enzyme, however, gave slightly higher product yields and could be used at higher ethanol concentrations without serious loss of activity.     

Trametes sp. LS-10C固态发酵产漆酶培养基优化及其对双酚A的降解

漆酶是一种绿色高效的多酚氧化酶,在降解双酚A方面具有巨大潜力。为降低发酵产漆酶的成本及考察漆酶在双酚A降解中的能力,本研究以麸皮和柚皮为主要基质,优化了栓菌固态发酵产漆酶条件,对优化后获得的漆酶在双酚A降解中的应用进行了研究。结果表明,在培养基组分为：麸皮和柚皮粉比例为6:4（W/W）、固液比1:2.5（W/V）、铜离子2%（W/W）、蔗糖3%（W/W）、硝酸钾2%（W/W）、稻壳20%（W/W）的条件下,栓菌发酵产漆酶的酶活最高,发酵11d后,酶活可达到38.4U/gds。当双酚A初始浓度为10μg/mL时,在55℃条件下酶解140min后,双酚A基本降解完全。     

Synthesis and anticancer evaluation of certain indolo[2,3-b]quinoline derivatives

The present report describes the synthesis and anticancer evaluation of certain 11-substituted 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 6H-indolo[2,3-b]quinoline derivatives 11–13 were prepared from the commercially available 1,4-dihydroxyquinoline through alkylation, chlorination, nucleophilic reaction, and ring cyclization. Depending on the ratio of 11, (MeO)₂SO₂, and K₂CO₃, alkylation occurred primarily on N-5 (1:0.8:0.8) or N-6 (1:1.5:1.5) leading to the isolation of 14a or 14b as a major product. Accordingly, major product 15a (2/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 15b (1:1:1), respectively, was obtained by alkylation of 12 while 16a (13/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 16b (1:1:1), respectively, was obtained by alkylation of 13. The in vitro anticancer assay indicated 5-methylated derivatives 14a, 15a, 16a are more cytotoxic than their respective 6-methylated counterparts 14b, 15b, 16b and 6H-indolo[2,3-b]quinoline precursors 11, 12, 13. Among them, 11-(4-methoxyanilino)-6-methyl-6H-indolo[2,3-b]quinoline (16a) was the most cytotoxic with a mean GI₅₀ value of 0.78 μM and also exhibited selective cytotoxicities for HL-60 (TB), K-562, MOLT-4, RPMI-8226, and SR with GI₅₀ values of 0.11, 0.42, 0.09, 0.14, and 0.19 μM, respectively.     

Angeli's salt and spinal motor neuron injury

Nitroxyl anion or its conjugate acid (NO_-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied.

Results: Freshly prepared AS (5 or 10 μmol), but not papanonoate, caused a marked decrease in the rotarod performance 3-7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5 μmol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons.

Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.     

Biotransformation of β-hydroxyphenyl selenides, diphenyldiselenide and benzeneseleninic acid by whole cells of Aspergillus terreus

Aspergillus terreus CCT 3320 and A. terreus URM 3571 catalysed the biotransformations of organic β-hydroxyphenyl selenides through oxidation and methylation reactions. The kinetic resolution of (RS)-1-(phenylseleno)-2-propanol (1) via enantioselective oxidation produced (+)-(S)-1 in high enantiomeric excess (>99%) and in a yield of 50% as determined by product isolation. Oxidation of the R-enantiomer of 1, followed by elimination of the propyl moiety and subsequent methylation of the presumed intermediate, led to the formation of methylphenyl-selenide, which was isolated in a yield of 40%. Whole cells of A. terreus also biocatalysed transformations of diphenyldiselenide, benzeneseleninic acid, (RS)-1-(phenylseleno)-2-pentanol and (RS)-1-(phenylseleno)-3-methyl-2-butanol, but not of (RS)-1-(phenylseleno)-2-phenyl-methanol. This is the first report of the biomethylation of organoselenium compounds by whole cells of A. terreus.     

Poly-γ-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity

The Bacillus subtilis pgdS gene, which is located at the immediate downstream of the pgs operon for poly-γ-glutamate (PGA) biosynthesis, encodes a PGA depolymerase. The pgdS gene product shows the structural feature of a membrane-associated protein. The mature form of the gene product, identified as a B. subtilis extracellular protein, was produced in Escherichia coli clone cells. Since the mature PGA depolymerase has been modified with the histidine-tag at its C-terminus, it could be simply purified by metal-chelating affinity chromatography. This purified enzyme digested PGAs from B. subtilis ( -glutamate content, 70%) and from Bacillus megaterium (30%) in an endopeptidase-like fashion. In contrast, PGA from Natrialba aegyptiaca, which consists only of -glutamate, was resistant to the enzyme, suggesting that, unlike fungal PGA endo-depolymerases, the bacterial enzyme recognizes the -glutamate unit in PGA.