Studies on the interaction of porphyrin with antibody with fluorescent spectrum and UV-Vis differential spectra

After meso-tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin combined with McAb 1F2, there was a significant hyperchromic effect, indicating that the combination of porphyrin and antibody is rigid and compact, aromatic amino acids exist at the combining sites of antigen in antibody. These aromatic amino acids are Trys and Trps, but the numbers of Trp are more than that found for Trys. The stochiometric ratio of porphyrin to 1F2 is 1:1, the disassociation constant was determined as(2.084+/-0.216) x 10(-10) mol/L by a method of fluorescence quenching, showing that both have a high affinity.

Bright blue electroluminescent devices utilizing poly (N-vinylcarbazole) doped with fluorescent dye

Bright blue electroluminescent devices have been fabricated using poly (N-vinylcarbazole) (PVK) doped with perylene as the emissive layer, poly(p-phenylenevinylene) (PPV) as the hole-transporting layer, 2-(4-biphenylyl)-5-(4-tert-butylphenyl)-1,3,4-oxadiazole (PBD), tris(8-hydroxyquinoline)aluminum (Alq(3)) as the electron-transporting layer, and Al as the cathode. A luminance of 700 cd/m(2) and a luminescent efficiency of 0.8% are achieved at a drive voltage of 36 V. In the experiment, it is found that the introduction of electron-transporting layer PBD has a great effect on the emissive color of the electroluminescent devices prepared by PVK doped with perylene. Yellow-green emission is observed from the device structure of glass substrate/indium-tin-oxide/PVK:perylene/Al. The possible emissive mechanisms are given. The effect of the transporting layer on the electroluminescence is also discussed. (C) 1997 Elsevier Science S.A.

SYNTHESIS OF THE CLINOPYROXENES JOIN CAMGSI2O6-NAALSI2O6 FOR JEWELRY

Clinopyroxenes of the join CaMgSi2O6(diopside)-NaAlSi2O6 (jadeite) were synthesized in the temperature range 800-1900 degrees C and under varying pressure, 10-55 kbar. The stability regions of various compositions of diopside-jadeite have been established experimentally using different compositions of glass materials: stoichiometric composition NaAlSi2O6, 0.1CaMgSi(2)O(6)-0.9NaAlSi(2)O(6), 0.2CaMgSi(2)O(6)0.8NaAlSi(2)O(6), 0.3CaMgSi(2)O(6)-0.7NaAlSi(2)O(6), and 0.4CaMgSi(2)O(6)-0.6NaAlSi(2)O(6). Unit cell parameters of synthetic clinopyroxenes with the above compositions were determined. The physical properties, such as hardness, toughness, density, and refractive index, etc., were also measured. The results show that synthetic clinopyroxenes have the same properties as the natural one. The gem quality of diopside-jadeite clinopyroxenes was achieved by synthesised on the basis of the above experiments. Various colouring agents, such as Cr2O3, Co2O3, NiO2, Fe2O3, TiO2, MnO, CuO, and their combinations, FeO-Cr2O3, etc., were added to obtain the different colours of gem. In addition, small amounts of the rare-earth oxides, such as CeO2, Nd2O3, Sm2O3, Dy2O3, Eu2O3, Er2O3, Pr6O11, Lu2O3 and CuO-Eu2O3, Co2O3Nd2O3, etc., were also added to produce fluorescent clinopyroxenes for jewellery.

A potent anti-oxidant property: fluorescent recombinant alpha-phycocyanin of Spirulina

To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni2+ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC50 values of rHHPC were 277.5 +/- 25.8 mu g ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 mu g ml(-1) against peroxyl radicals. Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximate to 20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Highly soluble and stable recombinant holo-phycocyanin alpha subunit expressed in Escherichia coli

C-phycocyanin (Cpc) is one of the phycobiliproteins with highly fluorescent and various pharmacological activities Holo-Cpc-alpha Subunit (holo-CpcA) expressed in Escherichia colt resulted in low yield and tended to aggregate after purification in this Study, we constructed a new plasmid coding holo-CpcA fused with hexahistidine and maltose-binding protein tag, which designated as HMCpcA. to Improve Its Solubility and stability without the Impairment of its spectra anti fluorescent properties HMCpcA was significantly more stable over time and a wider range of pH as compared to holo-CpcA. In addition. both the solubility and yields of HMCpcA increase significantly We here provided an example to demonstrate that MBP could also Improve the stability of the protein it fused while it has been reported as a soluble fusion partner before. This novel fluorescent protein will facilitate the large-scale production and be potentially applicable for the development Of fluorescent probes, as well as antioxidant agents (C) 2009 Elsevier B V. All rights reserved

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be auto-catalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases (HOI (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC50 values were 296.4 +/- 22.4 mu g/ml against hydroxyl radicals and 38.5 +/- 2.6 mu g/ml against peroxyl radicals.

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Synthesis and QSAR studies of novel triazole compounds containing thioamide as potential antifungal agents

Eighteen novel triazole compounds containing thioamide were designed and synthesized. Their structures were confirmed by elemental analysis, H-1 NMR, IR, and MS. The title compounds exhibited certain antifungal activity. And the geometry structures of the title compounds were optimized by means of the density functional theory (DFT) method at B3LYP/6-31G* level. The quantitative structure-activity relationship (QSAR) of the title compounds was systematically investigated. A correlative equation between FA and DELH, V was well established by using the multiple linear regression (MLR). (c) 2006 Elsevier Ltd. All rights reserved.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

A novel method to synthesize amorphous silica-alumina materials with mesoporous distribution without using templates and pore-regulating agents

In this study, a novel sol-gel method is used to synthesize amorphous silica-alumina materials with a narrow mesoporous distribution and various Si/Al molar ratios without using any templates and pore-regulating agents. During the preparation procedure, only inexpensive inorganic salts were used as raw materials, instead of expensive and harmful alkoxides. The precursor sol was dried at room temperature in a vacuum box kept at 60 mmHg until it began to form the gel. The results of a nitrogen sorption experiment indicate that the synthesized materials with different Si/Al molar ratios have similar mesoporous distributions (within 2-12 nm). Moreover, it was found that the material's pore size distribution remains at a similar value during the heat treatment from room temperature to 550 degreesC. On the basis of the nitrogen sorption, TEM, and AFM characterization results, a formation mechanism of mesopores which accounts for the experimental data is also suggested. This suggested mechanism involves rearrangement of the primary particles during the drying process to form the precursors of the similarly sized mesopores. The synthesized materials were characterized by XRD, thermal analysis (TG/DTA), Al-27 and Si-29 MAS NMR spectroscopy, SEM, TEM, and AFM. The results of Al-27 and 29Si MAS NMR indicate that the distribution of silicon and aluminum in the synthesized materials is more uniform and homogeneous than that in the mixed oxides prepared via the traditional sol-gel method even at high alumina contents. The type and density of the acid sites were studied using pyridine adsorption-desorption FTIR spectroscopy. It was shown that the acidity of the synthesized materials is higher than that of the silica-alumina materials prepared by conventional methods.

Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay

A new nonadentate ligand, N, N, N-1, N-1-[2,6-bis(3'-aminomethyl-1 1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb3+ fluorescent complex was synthesized. The Tb3+ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifetime of 2.681 ms in 0.05 M berate buffer of pH 9.1. Streptavidin (SA) was labeled with SPTA by using its succinimidyl monoester, and the BPTA-Tb3+-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of alpha -fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb3+-labeled SA was also used in competitive type TR-FIA of bensulfuron- methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simultaneous measurement method for AFP and CEA in a human serum sample was developed by using 4,4'-bis(1 " ,1 " ,1 " ,2 " ,2 " ,3 " ,3 " -heptafluoro-4 " ,6 " -hexanedion-6 " -yl)chlorosulfo-o-terphenyl ((BHHCT)-Eu3+-labeled anti-AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled SA. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.