6-Pyruvoyltetrahydropterin synthase orthologs of either a single or dual domain structure are responsible for tetrahydrobiopterin synthesis in bacteria

6-Pyruvoyltetrahydropterin synthase (PTPS) catalyzes the second step of tetrahydrobiopterin (BH4) synthesis. We previously identified PTPS orthologs (bPTPS-Is) in bacteria which do not produce BH4. In this study we disrupted the gene encoding bPTPS-I in Synechococcus sp. PCC 7942, which produces BH4-glucoside. The mutant was normal in BH4-glucoside production, demonstrating that bPTPS-I does not participate in BH4 synthesis in vivo and bringing us a new PTPS ortholog (bPTPS-II) of a bimodular polypeptide. The recombinant Synechococcus bPTPS-II was assayed in vitro to show PTPS activity higher than human enzyme. Further computational analysis revealed the presence of mono and bimodular bPTPS-II orthologs mostly in green sulfur bacteria and cyanobacteria, respectively, which are well known for BH4-glycoside production. In summary we found new bacterial PTPS orthologs, having either a single or dual domain structure and being responsible for BH4 synthesis in vivo, thereby disclosing all the bacterial PTPS homologs.     

Green synthesis and characterization of zinc oxide nanoparticles using Cayratia pedata leaf extract

The synthesis of Zinc oxide nanoparticles using a plant-mediated approach is presented in this paper. The nanoparticles were successfully synthesized using the Nitrate derivative of Zinc and plant extract of the indigenous medicinal plant Cayratia pedata. 0.1 mM of Zn (NO₃)₂.6H₂O was made to react with the plant extract at different concentrations, and the reaction temperature was maintained at 55 °C, 65 °C, and 75 °C. The yellow coloured paste obtained was wholly dried, collected, and packed for further analysis. In the UV visible spectrometer (UV–Vis) absorption peak was observed at 320 nm, which is specific for Zinc oxide nanoparticles. The characterization carried out using Field Emission Scanning Electron Microscope (FESEM) reveals the presence of Zinc oxide nanoparticles in its agglomerated form. From the X-ray diffraction (XRD) pattern, the average size of the nanoparticles was estimated to be 52.24 nm. Energy Dispersive Spectrum (EDX) results show the composition of Zinc and Oxygen, giving strong energy signals of 78.32% and 12.78% for Zinc and Oxygen, respectively. Fourier Transform - Infra-Red (FT-IR) spectroscopic analysis shows absorption peak of Zn–O bonding between 400 and 600 cm^?1. The various characterization methods carried out confirm the formation of nano Zinc oxide. The synthesized nanoparticles were used in the immobilization of the enzyme Glucose oxidase. Relative activity of 60% was obtained when Glucose oxidase was immobilized with the green synthesized ZnO nanoparticles. A comparative study of the green synthesized with native ZnO was also carried out. This green method of synthesis was found to be cost-effective and eco-friendly.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris

The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of ³²P-labeled sphingosine 1-phosphate.     

Spike‐triggered dendritic calcium transients depend on synaptic activity in the cricket giant interneurons

The relationship between electrical activity and spike‐induced Ca²⁺ increases in dendrites was investigated in the identified wind‐sensitive giant interneurons in the cricket. We applied a high‐speed Ca²⁺ imaging technique to the giant interneurons, and succeeded in recording the transient Ca²⁺ increases (Ca²⁺ transients) induced by a single action potential, which was evoked by presynaptic stimulus to the sensory neurons. The dendritic Ca²⁺ transients evoked by a pair of action potentials accumulated when spike intervals were shorter than 100 ms. The amplitude of the Ca²⁺ transients induced by a train of spikes depended on the number of action potentials. When stimulation pulses evoking the same numbers of action potentials were separately applied to the ipsi‐ or contra‐lateral cercal sensory nerves, the dendritic Ca²⁺ transients induced by these presynaptic stimuli were different in their amplitude. Furthermore, the side of presynaptic stimulation that evoked larger Ca²⁺ transients depended on the location of the recorded dendritic regions. This result means that the spike‐triggered Ca²⁺ transients in dendrites depend on postsynaptic activity. It is proposed that Ca²⁺ entry through voltage‐dependent Ca²⁺ channels activated by the action potentials will be enhanced by excitatory synaptic inputs at the dendrites in the cricket giant interneurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 234–244, 2002; DOI 10.1002/neu.10032     

Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose

Endophytic fungi belonging to the genus Gliocladium are able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. These fungi could thus be used to produce biofuels from cellulosics without the need for hydrolytic pretreatments. Gas chromatography-mass spectrometry-solid-phase micro-extraction (GC-MS-SPME) of head space gases from Gliocladium cultures demonstrated the production of C(6)-C(19) hydrocarbons including hexane, benzene, heptane, 3,4-dimethyl hexane, 1-octene, m-xylene, 3-methyl nonane, dodecane, tridecane, hexadecane and nonadecane directly from the cellulosic biomass. Hydrocarbon production was 100-fold higher in co-cultures of Gliocladium and Escherichia coli than in pure Gliocladium cultures. The dry mycelia weight is stable at stationary period in co-culture condition which may lead to synthesize more hydrocarbons. These fungi could potentially be developed into cost-effective biocatalysts for production of biofuels.     

绿色荧光报告基因在细胞免疫检测中的应用

将丙肝病毒C E1区基因插入绿色荧光报告基因pEGFP-N1中,构建真核表达重组质粒pEGFP-N1-HCV/C E1。转染小鼠骨髓瘤细胞SP2/0,在荧光显微镜下观察绿色荧光融合蛋白的表达情况。结果在细胞浆中出现了绿色荧光,表明目的基因得到表达,再通过G418筛选后大量培养用作细胞毒实验的靶细胞,结果表明以EGFP报告基因作筛选标记制备的靶细胞完全可以满足细胞毒实验要求。     

Kinetic studies on the diaqua form of cis-platin and various nucleobases

Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity.     

A convenient ‘one-pot’ synthesis and in vitro microbiological evaluation of novel 2,7-diaryl-[1,4]-diazepan-5-ones

A convenient method for the ‘one-pot’ synthesis of novel target molecule 2,7-diaryl-[1,4]-diazepan-5-ones from the respective 2,6-diaryl-piperidin-4-ones was catalyzed by NaHSO₄.Al₂O₃ heterogeneous catalyst in dry media under microwave irradiation in solvent-free conditions. Moreover, the catalyst could be recovered and re-used up to 4 times after washing with ethyl acetate. They were evaluated for potential antibacterial activity against Staphylococcus aureus, β-Haemolytic streptococcus, Vibreo cholerae, Salmonella typhii, Escherichia coli, Klebsiella pneumonia, Pseudomonas and antifungal activity against Aspergillus flavus, Aspergillus fumigatus, Mucor, Candida albicans and Rhizopus. Structure-Activity Relationship (SAR) led to the conclusion that, of all the compounds 25–32 tested, compound 30 exerted strong in vitro antibacterial activity against S. aureus, S. typhii, and Pseudomonas and all the compounds 25–32 were less active against E. coli, whereas all the compounds 25–32 displayed potent in vitro antifungal activity against all the fungal strains used, except compound 30, which was more effectual against Mucor.