Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12

Summary Insertion of the fusion-generating phage Mud1 (Ap, lacZ) yielded two similar isolates, DC511 and DC512, which were unable to grow aerobically on acetate or alphaketoglutarate but which could use succinate, malate, fumarate, glycerol, and various sugars. These mutants were unable to grow anaerobically on most sugars unless provided with methionine, lysine, and delta-aminolevulinic acid, all of which require succinyl-CoA for their synthesis. The insertions of both mutants mapped at 17 min, in the suc operon. Enzyme assays indicated a lack of succinyl-CoA synthetase; however, full activity of the alpha-ketoglutarate dehydrogenase was retained. Beta-galactosidase expression by strains containing these gene fusions was reduced under anaerobic conditions. In aerobically grown cultures, both fusions were induced about fivefold in the presence of acetate. This type of regulation would be expected of a Krebs cycle enzyme.     

Ascaris lumbricoides: succinate and tiglate in hemolymph

When hemolymph is taken from Ascaris lumbricoides at the time the worm is collected from pigs, it contains acetic, propionic, 2-methylbutyric, n-valeric, 2-methylvaleric, and succinic acid radicals; tiglic acid is absent.     

Effect of carbamate herbicides on VA mycorrhizal infection and plant growth

Summary The effect of the carbamate herbicides Chlorpropham, Sulfallate and Phenmedipham, which are believed to inhibit photosynthesis, on VA mycorrhizal infection and on plant growth, were examined. Foliar spraying of Phenmedipham decreased the root concentration of total and reducing sugars and the fungal metabolism (using a staining reaction for succinate dehydrogenase as indicator) 48 h after application. However, all three carbamate herbicides tested, whether applied by foliar spray or directly to soil, did not affect the amount of VA mycorrhizal infection present at the end of the experiment. These herbicides decreased plant growth when they were applied to soil. But when the herbicides were sprayed only Phenmedipham, applied at high concentrations, decreased plant growth. Moreover, our results show that VA mycorrhizas may help plants recover from the deleterious effect of Phenmedipham.     

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using ¹³C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1 mol mol⁻¹ and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71 g L⁻¹ succinate and a yield of 2.5 mol mol⁻¹ from sucrose.     

In vitro culture of human chondrocytes (1): A novel enhancement action of ferrous sulfate on the differentiation of human chondrocytes

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO₄) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO₄ on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO₄, or both bFGF + FeSO₄ for4weeks. A 20 μl aliquot of a cell suspension containing2 × 10⁷ cells ml⁻¹ was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO₄, and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO₄,and bFGF + FeSO₄ were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO₄ andbFGF + FeSO₄. The combined effects of bFGF and FeSO₄ were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.     

Glutamate induces H2O2 synthesis in nonsynaptic brain mitochondria

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H₂O₂ formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H₂O₂ generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca²⁺ (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5 mM). We have shown that the H₂O₂ formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H₂O₂ formation through mitochondrial respiratory chain complex II. The H₂O₂ formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.     

Design,synthesis, fungicidal activity and molecular docking studies of novel 2-((2-hydroxyphenyl)methylamino)acetamide derivatives

A series of novel 2-hydroxyphenyl substituted aminoacetamides was designed by molecular hybridization of the aminoacetamide scaffold and 2-hydroxyphenyl motif. The target compounds were synthesized and their fungicidal activities were evaluated. Some of the target compounds showed excellent antifungal activities against S. sclerotiorum and P. capsici. Significantly, compounds 5e displayed the most potent activity against S. sclerotiorum with EC₅₀ = 2.89 µg/mL, which was lower than that of commercial chlorothalonil. The systematic studies provided strong confidence that the hydroxyl group and the carbonyl group are crucial for the fungicidal activity. Molecular docking studies suggest that SDH enzyme could be one of the potential action targets of our compounds.     

复方黄连素片中盐酸小檗碱含量测定

目的：建立测试黄连素片中盐酸小檗碱含量的高效液相方法,并对此方法进行系统的方法学验证,以确保应用该方法测试的结果准确、可靠。方法：采用色谱柱：Agilent TC-C18柱（4．6 mm ×150 mm,5μm,Agilent,美国）,流动相：乙腈－0．05 mol／L磷酸二氢钾溶液（45∶55,含5％的0．05 mol／L庚烷磺酸钠溶液）,检测波长为265 nm。结果：盐酸小檗碱在2．5～80μg／mL范围内线性关系良好,得到线性回归方程为Y＝421 ．5X＋17．32,相关系数为0．9997（n＝6）;低、中、高浓度批内精密度的RSD值分别为0．30％、0．58％、0．30％,批间精密度的RSD为0．80％;重复性RSD为0．27％;低、中、高浓度的加样回收率分别为99．66％、99．88％、99．98％;供试品溶液室温放置8h稳定,RSD为0．41％。结论：本方法测定黄连素片中盐酸小檗碱的含量准确、可靠,操作简单、用时短,可用于黄连素片中盐酸小檗碱的含量测定。