Study on preventive and therapeutic effect of Tongbu-fangchan prescription on Ach-CaCl / 中国药理学通报

Aim To explore he preventive and therapeutic effects of Tongbu-fangchan prescription on aceylcholine-calcium chloride (Ach-CaCl

Fluorescent intracellular imaging of reactive oxygen species and pH levels moderated by a hydrogenase mimic in living cells / 药物分析学报

The catalytic generation of H2 in living cells provides a method for antioxidant therapy.In this study,an[FeFe]-hydrogenase mimic[Ru+Fe2S2@F127(80)]was synthesized by self-assembling polymeric plur-onic F-127,catalytic[Fe2S2]sites,and photosensitizer Ru(bpy)32+.Under blue light irradiation,hydrated protons were photochemically reduced to H2,which increased the local pH in living cells(HeLa cells).The generated H2 was subsequently used as an antioxidant to decrease reactive oxygen species(ROS)levels in living cells(HEK 293T,HepG2,MCF-7,and HeLa cells).Our findings revealed that the proliferation of HEK 293T cells increased by a factor of about six times,relative to that of other cells(HepG2,MCF-7,and HeLa cells).Intracellular ROS and pH levels were then monitored using fluorescent cell imaging.Our study showed that cell imaging can be used to evaluate the ability of Ru+Fe2S2@F127 to eliminate oxidative stress and prevent ROS-related diseases.

Knockdown of IOP1 Induces Premature Senescence in Primary HUVECs / 中国生物化学与分子生物学报

Iron-only hydrogenase-like protein 1 (IOP1) is a component of the cytosolic iron-sulfur protein assembly (CIA) machinery. IOP1 has been suggested to be a negative regulator of the hypoxia-inducible transcription factor 1(HIF-1). We previously reported that loss of one copy of NAR1 (the yeast homolog of IOP1) in diploid yeast cells leads to increased sensitivity to oxidative stress and decreased replicative lifespan‚ however‚ the underlying mechanism is still unclear. Recently‚ we found that the IOP1 protein was upregulated in late-passaged primary human umbilical vein endothelial cells (HUVECs) compared with that in early-passaged primary HUVECs‚ which indicated a potential association of IOP1 with cellular senescence. The aim of this study was to investigate the potential function of IOP1 in aging in mammalian cells. The primary HUVECs were transfected with IOP1-specific siRNA and subjected to premature senescence assays. We found that IOP1 knockdown leads to premature senescence and decreased cell proliferative ability (P < 0. 01) in primary HUVECs. Further studies revealed that downregulation of IOP1 resulted in upregulated ROS levels (P < 0. 01)‚ enhanced DNA damage (P<0. 05) and decreased mitochondrial respiration (P<0. 01) along with cell cycle arrest at the G

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

Background: The development of a potential single culture that can co-produce hydrogen and ethanol is beneficial for industrial application. Strain improvement via molecular approach was proposed on hydrogen and ethanol co-producing bacterium, Escherichia coli SS1. Thus, the effect of additional copy of native hydrogenase gene hybC on hydrogen and ethanol co-production by E. coli SS1 was investigated. Results: Both E. coli SS1 and the recombinant hybC were subjected to fermentation using 10 g/L of glycerol at initial pH 7.5. Recombinant hybC had about 2-fold higher cell growth, 5.2-fold higher glycerol consumption rate and 3-fold higher ethanol productivity in comparison to wild-type SS1. Nevertheless, wild-type SS1 reported hydrogen yield of 0.57 mol/mol glycerol and ethanol yield of 0.88 mol/mol glycerol, which were 4- and 1.4-fold higher in comparison to recombinant hybC. Glucose fermentation was also conducted for comparison study. The performance of wild-type SS1 and recombinant hybC showed relatively similar results during glucose fermentation. Additional copy of hybC gene could manipulate the glycerol metabolic pathway of E. coli SS1 under slightly alkaline condition. Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

Background: An effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated. Results: Recombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1. Conclusions: Insertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia.

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under freeliving conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).

Hydrogenosomal activity of Trichomonas vaginalis cultivated under different iron conditions

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.

EFFECT OF DIFFERENT PHOSPHATE BUFFER CONCENTRATIONS ON HYDROGENASE ACTIVITY AND HYDROGEN PRODUCTION OF CLOSTRIDIUM BUTYRICUM A69 / 微生物学通报

Clostridium butyricum was incubated in 10m mol, 30m mol 50m mol and 70m mol phosphate buffer medium (pH 7.0). With the high concentration buffer, the pH value decreased more slowly. The H_2 evolution hydrogenase kept on high activity level for a longer time, While the H_2 uptake hydrogenase kept constant. So more hydrogen was produced. The hydrogen production nearly doubled in 70m mol phosphate buffer than in 10m tool phosphate buffer. The result shows that to keep pH constant can increase the H_2 production of C. butyricum.

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany.

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup+ (S24 and CT2014) and one Hup– (M11) Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H2 uptake rates were higher in 1 and 2% H2 in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H2 uptake were observed both, due to host species as well as due to Rhizobium strains. However, no host dependent complete repression of the expression of H2 uptake activity was observed in nodules of any of the host species formed with Hup+ strains.