RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer.

Chemotherapeutic agents targeting energy metabolism have not achieved satisfactory results in different types of tumors. Herein, we developed an RNA interference (RNAi) method against adenosine triphosphate (ATP) by constructing an interfering plasmid-expressing ATP-binding RNA aptamer, which notably inhibited the growth of prostate cancer cells through diminishing the availability of cytoplasmic ATP and impairing the homeostasis of energy metabolism, and both glycolysis and oxidative phosphorylation were suppressed after RNAi treatment. Further identifying the mechanism underlying the effects of ATP aptamer, we surprisingly found that it markedly reduced the activity of membrane ionic channels and membrane potential which led to the dysfunction of mitochondria, such as the decrease of mitochondrial number, reduction in the respiration rate, and decline of mitochondrial membrane potential and ATP production. Meanwhile, the shortage of ATP impeded the formation of lamellipodia that are essential for the movement of cells, consequently resulting in a significant reduction of cell migration. Both the downregulation of the phosphorylation of AMP-activated protein kinase (AMPK) and endoplasmic reticulum kinase (ERK) and diminishing of lamellipodium formation led to cell apoptosis as well as the inhibition of angiogenesis and invasion. In conclusion, as the first RNAi modality targeting the blocking of ATP consumption, the present method can disturb the respiratory chain and ATP pool, which provides a novel regime for tumor therapies..

Synthesis and Evaluation of Phenylxanthine Derivatives as Potential Dual A2AR Antagonists/MAO-B Inhibitors for Parkinson's Disease.

The aim of this research was to prove the speculation that phenylxanthine (PX) derivatives possess adenosine A2A receptor (A2AR)-blocking properties and to screening and evaluate these PX derivatives as dual A2AR antagonists/MAO-B inhibitors for Parkinson's disease. To explore this hypothesis, two series of PX derivatives were prepared and their antagonism against A2AR and inhibition against MAO-B were determined in vitro. In order to evaluate further the antiparkinsonian properties, pharmacokinetic and haloperidol-induced catalepsy experiments were carried out in vivo. The PX-D and PX-E analogues acted as potent A2AR antagonists with Ki values ranging from 0.27 to 10 µM, and these analogues displayed relatively mild MAO-B inhibition potencies, with inhibitor dissociation constants (Ki values) ranging from 0.25 to 10 µM. Further, the compounds PX-D-P6 and PX-E-P8 displayed efficacious antiparkinsonian properties in haloperidol-induced catalepsy experiments, verifying that these two compounds were potent A2AR antagonists and MAO-B inhibitors. We conclude that PX-D and PX-E analogues are a promising candidate class of dual-acting compounds for treating Parkinson's disease.

[Species identification of grouper and snapper in Taiwan Strait using polymerase chain reaction-restriction fragment length polymorphism analysis and lab-on-a-chip system].

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a-chip system were used to identify grouper and snapper species in Taiwan Strait. A fragment of 464 bp length of mitochondrial cytochrome b gene was amplified by PCR and the products were digested with restriction enzymes Dde I , Hae III and NLa III, individually. The fragments generated after digestion were further resolved on the DNA Chip. Eight grouper species and five snapper species were successfully identified. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provide a fast, easy, automated, and reliable analysis approach. This approach is potential for the purpose of fish adulteration control.

Saccharofermentans acetigenes gen. nov., sp. nov., an anaerobic bacterium isolated from sludge treating brewery wastewater.

A spore-forming anaerobic bacterium, designated strain P6(T), was isolated from the sludge of an up-flow anaerobic sludge blanket reactor treating brewery wastewater. Cells were Gram-positive, oval and 0.6-0.9 µm by 1.2-1.8 µm in size. Growth was observed at 20-42 °C and at pH 5.0-7.5. It fermented several hexoses, polysaccharides and alcohols. Sucrose and aesculin could also be fermented. The main end products of fermentation from glucose were acetate, lactate and fumarate; trace CO(2) and H(2) were also produced. The DNA G+C content of strain P6(T) was 55.6 mol%. The major cellular fatty acids were iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(14 : 0) 3-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain P6(T) represented a novel phyletic sublineage in clostridial cluster III, and showed <91 % similarity to the type strains of recognized species in this cluster. Phenotypically, the new isolate was distinguished from its phylogenetic relatives (e.g. Clostridium straminisolvens, Clostridium thermocellum, Acetivibrio cellulolyticus and Clostridium aldrichii) by producing acid from glucose and its inability to degrade cellulose. On the basis of evidence from this polyphasic study, strain P6(T) is considered to represent a novel species of a new genus, for which the name Saccharofermentans acetigenes gen. nov., sp. nov. is proposed. The type strain of Saccharofermentans acetigenes is P6(T) (=JCM 14006(T) =AS 1.5064(T)).

[Hydrogen production from glucose by Acetanaerobacterium elongatum].

Studying the possible factors affecting hydrogen production from glucose by Acetanaerobacterium elongatum, an acetateethanol fermenting anaerobe isolated from sludge of wastewater of a papermill. The results revealed that the maximum hydrogen production could be achieved in PYG liquid at 37 degrees C and initial pH 8.0. The strain produced more hydrogen from glucose and arabiose, with the hydrogen yields of 1.55 mol H2/mol glucose and 1.50 mol H2/mol arabiose, respectively. pH and hydrogen partial pressure were the most severe inhibitors in hydrogen production from glucose, by controlling the broth at neutral pH during the fermentation period could increase hydrogen yield twice, concomitantly glucose consumption for 2.8 times. Hydrogen was another inhibitor for fermentative H2 production, and removing hydrogen by cultivating Acetanaerobacterium elongatum Z7 with a H2-trophic methanogen resulted in higher deduced H2 production for about 4 times. Yeast extract stimulated H2 production by stains Z7. Coculturing A. elongatum Z7 and P. acetatigenes TB107, a proteineous-trophic bacterium, increased the conversion of the glucose and thereby increased the H2 production on peptone. Moreover, acetate when at higher concentration ( > 60 mmol/L) in the medium inhibited the hydrogen production by strain Z7.

[A new hydrogen-producing strain and its characterization of hydrogen production].

An anaerobic, thermophilic, hydrogen-producing strain T42 was obtained from a hot spring of South Mountain District, Tibet. Cells are Gram-positive, mobile rod-shaped. Spores were not observed. Temperature range for growth is 32 degrees C to 69 degrees C (optimum temperature, 60 degrees C - 62 degrees C), and pH range for growth is 5.0 to 8.8 (optimum pH, 7.0 - 7.5). The generation time is around 30 min. Organic nitrogen sourc is required for growth. Strain T42 utilizes a wide range of carbohydrates, including starch, dextrin, sucrose, cellobiose, fructose, maltose, ribose, glycogen and galactose. Acetate, ethanol, H2 and CO2 are the end products of glucose fermentation. The (G + C) content of strain T42 is 31.2 mol%. Phylogenetic analysis based on the 16S rDNA sequence similarity indicates that strain T42 is the closest relative to Thermobrachium celere and Caloramator indicus. Biological characteristics and phylogenetic analysis of the 16S rDNA gene indicate the new strain belongs to the genus Thermobrachium. Strain T42 produces H2 from glucose at maximal level when growing at 62 degrees C and initial pH 7.2, the hydrogen yields and maximal hydrogen production rate are 1.06 mol H2/mol glucose and 24.0 mmol H2/gDW/h, respectively. Strain T42 also produced H2 by fermentating from a variety of carbohydrates. 20 mmol/L Magnesium and 2 mmol/L iron increase the hydrogen production content by 20% and 23.3%, respectively, but nickel has no effect on the hydrogen production. In the co-culture of strain T42 and methane-producing strain M. thermautotrophicus Z245, hydrogen pressure is dramatically decreased, meanwhile deduced H2 production and the consumption of glucose are increased markedly by 2.8 fold and 1 fold, and the ratio of acetate/ethanol is enhanced froml to 1.7.

Sporacetigenium mesophilum gen. nov., sp. nov., isolated from an anaerobic digester treating municipal solid waste and sewage.

Two mesophilic, anaerobic bacterial strains (ZLJ115T and L4-2) were isolated from the sludge of an anaerobic digester treating municipal solid waste and sewage in Fujian province, China. The strains were Gram-positive, spore-forming, motile rods (0.9-1.0 x 3.6-7.3 microm). Growth of the strains was observed at 20-42 degrees C and pH 6.0-9.5. Both strains fermented several mono- and disaccharides. The main fermentation products from glucose were acetate, ethanol, hydrogen and carbon dioxide. Optimal hydrogen production by the new isolates was observed at pH 8.8 and 39 degrees C, and 1.4 mol H2 was detected from fermentation of 1 mol glucose. The DNA G+C contents of strains ZLJ115T and L4-2 were 53.9 and 54.3 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates represented a novel phyletic sublineage within cluster XI of the clostridia, clustering with four thermophilic species, with <93.8 % 16S rRNA gene sequence similarity to previously described species. Phenotypically, the new isolates were distinguished from their phylogenetic relatives by growing mesophilically and by fermenting a variety of pentoses, as well as their higher genome DNA G+C content. On the basis of polyphasic evidence from this study, a novel genus and species are proposed, Sporacetigenium mesophilum gen. nov., sp. nov.; strain ZLJ115T (=DSM 16796T = AS 1.5019T) is the type strain of Sporacetigenium mesophilum.

Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater.

Two proteolytic, strictly anaerobic bacterial strains (TB107(T) and TB6-6) were isolated from the granule sludge of an upflow anaerobic sludge blanket reactor treating brewery wastewater. The strains were Gram-negative, non-spore-forming and motile. Cells were rod-shaped (0.6-0.9x1.9-2.2 microm). Growth of the strains was observed at 20-45 degrees C and pH 6.0-9.7. The strains were proteolytic. Yeast extract, peptone, pyruvate, glycine and l-arginine could be used as carbon and energy sources. Weak growth was also observed with tryptone, l-serine, l-threonine and l-alanine as carbon and energy sources. Both strains did not use any of the tested carbohydrates, alcohols and fatty acids except pyruvate. Acetic acid and NH3 were produced from yeast extract, peptone and l-arginine, and propionic acid was also produced from yeast extract. Pyruvate was converted to acetic acid and CO2. Gelatin was not hydrolysed. Indole and H2S were not produced. The two strains did not grow in medium containing 20 % bile. Addition of strain TB107T to a syntrophic propionate-degrading co-culture accelerated the propionate-degradation rate. The predominant cellular fatty acid was the branched-chain fatty acid anteiso-C(15 : 0) (46.21 %). The genomic DNA G+C contents of strains TB107T and TB6-6 were 46.6 and 48.9 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains represent a new phyletic sublineage within the Cytophaga-Flavobacterium-Bacteroides (CFB) group, with <91 % 16S rRNA gene sequence similarity to the closest species with validly published names. On the basis of polyphasic evidence from this study, a new genus and species, Proteiniphilum acetatigenes gen. nov., sp. nov., is proposed, with strain TB107T (=JCM 12891T=AS 1.5024T) as the type strain.

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008.

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

Syntrophobacter sulfatireducens sp. nov., a novel syntrophic, propionate-oxidizing bacterium isolated from UASB reactors.

Two obligate anaerobes, TB8106(T) and WZH410, which degraded propionate in syntrophic association with methanogens, were isolated from two upflow anaerobic sludge blanket reactors, one treating brewery wastewater and the other bean curd wastewater. The strains were Gram-negative, non-spore-forming and non-motile. Cells were egg-shaped, with a size of 1.0-1.3 x 1.8-2.2 microm. Growth was observed at 20-48 degrees C and pH 6.2-8.8. Both strains converted propionate to acetate and methane in co-culture with methanogens, and grew on propionate plus sulfate in pure culture, with a doubling time of 52-55 h at 37 degrees C. Sulfate and thiosulfate both served as electron acceptors for propionate degradation. The DNA G + C contents of the two strains were 58.5 and 58.7 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to a propionate-oxidizing syntrophic bacterium, Syntrophobacter fumaroxidans DSM 10017(T) (94.7 % similarity). However, the novel strains could not ferment fumarate, and grew at a more alkaline pH range than Syntrophobacter fumaroxidans. Moreover, the novel isolates had obviously higher growth rates on propionate plus sulfate (0.12 day(-1)) than Syntrophobacter fumaroxidans DSM 10017(T) (0.024 day(-1)). Therefore, a novel species, Syntrophobacter sulfatireducens sp. nov., is proposed, with strain TB8106(T) (=AS 1.5016(T) = DSM 16706(T)) as the type strain.

Acetanaerobacterium elongatum gen. nov., sp. nov., from paper mill waste water.

Two mesophilic anaerobic bacterial strains (Z7(T) and Z1) were isolated from waste water sludge of the Xinanzhang paper mill, Beijing, China. The strains were Gram-positive, non-spore-forming and motile. Cells were thin rods (0.2-0.4x4.0-8.0 microm). Growth of the strains was observed at 20-42 degrees C and pH 5.0-7.5. Both strains hydrolysed gelatin and aesculin and fermented several kinds of mono-, di- and oligosaccharides. The fermentation end products formed from glucose were acetate, ethanol, hydrogen and carbon dioxide. The predominant cellular fatty acids were the branched-chain fatty acids isoC(15 : 0) (42.83 %) and isoC(14 : 0) (32.11 %). The DNA G+C contents of strains Z7(T) and Z1 were 50.4 and 48.6 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates represent a new phyletic sublineage within the Clostridium leptum rRNA cluster, with <91 % 16S rRNA gene sequence similarity to currently described species. On the basis of polyphasic evidence from this study, Acetanaerobacterium elongatum gen. nov., sp. nov., a novel genus and species, is proposed, with strain Z7(T) (=JCM 12359(T)=AS 1.5012(T)) as the type strain.