Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.

Phylogenetic and gene expression analysis of cyanobacteria and diatoms in the twilight waters of the temperate northeast Pacific Ocean.

In this study, to explore the microbial community structure and its functionality in the deep-sea environments, we initially performed a 16S ribosomal RNA (rRNA)-based community structure analyses for microbial communities in the sea water collected from sites of 765-790 m in depth in the Pacific Ocean. Interestingly, in the clone library we detected the presence of both photoautotrophic bacteria such as cyanobacteria and photoheterotrophic bacteria, such as Chloroflexus sp. To further explore the existence and diversity of possible light-utilizing microorganisms, we then constructed and analyzed a 23S rRNA plastid gene cloning library. The results showed that the majority of this cloning library was occupied by oxygenic photoautotrophic organisms, such as diatoms Thalassiosira spp. and cyanobacterium Synechococcus sp. In addition, the diversity of these oxygenic photoautotrophic organisms was very limited. Moreover, both reverse-transcription PCR and quantitative reverse-transcription PCR approaches had been employed to detect expression of the genes involved in protein synthesis and photosynthesis of photoautotrophic organisms, and the positive results were obtained. The possible mechanisms underlying the existence of very limited diversity of photosynthetic organisms at this depth of ocean, as well as the positive detection of rRNA and mRNA of diatom and cyanobacteria, were discussed.

Tracking bacterial infection of macrophages using a novel red-emission pH sensor.

The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria.

Multi-walled carbon nanotube-based carbon/carbon composites with three-dimensional network structures.

Multi-walled carbon nanotube (MWCNT)-based carbon/carbon composites were fabricated by the chemical vapor infiltration of pyrolytic carbon into pre-compressed MWCNT blocks. The pyrolytic carbon was deposited on the surface of the MWCNTs and filled the gaps between the MWCNTs, which improved the connection between the MWCNTs and formed a three-dimensional network structure. The mechanical and electrical properties were improved significantly. The values of the maximum compressed deformation, maximum breaking strength, Young's modulus and energy absorption are measured as 10.9%, 148.6 MPa, 1588.6 MPa and 13.8 kJ kg(-1), respectively. The conductivity reached about 204.4 S cm(-1), more than 10 times larger than that of pre-compressed MWCNT blocks. After annealing at 1800 °C in vacuum, the graphitization improved remarkably. The pyrolytic carbon deposited on the surface of the MWCNTs was rearranged along the walls, and resulted in an increase of the number of walls of the MWCNTs.

New ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activity in Cyanobacteria.

Photosynthetic algae and cyanobacteria have been proposed for producing biofuels through a direct photoconversion process. To accelerate the efforts of discovering and screening microbes for biofuel production, sensitive and high throughput methods to measure photosynthetic activity need to be developed. Here we report the development of new ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activities directly. The dual sensor system can measure oxygen (O(2)) generation and pH increase resulted from carbon dioxide (CO(2)) consumption simultaneously. The sensor was prepared by a copolymerization of three monomeric probes, an intra-reference probe (IRP) which does not respond to pH or O(2), a probe for pH sensing (pHS), and an O(2) probe for O(2) sensing (OS) with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM). After polymerization, the three probes were chemically immobilized in an ion and O(2) permeable poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrix. The resulted sensing films (membranes) exhibited three emission colors with well separated emission spectra, covering blue, green, and red emission windows, under 380 nm light excitation. Responses of the sensors to pH and dissolved O(2) were investigated in buffers and cyanobacterial cell cultures (Synechocystis sp. PCC 6803). In spite of the strong autofluorescence from cyanobacteria, the sensors were able to determine the pH values and dissolved O(2) concentrations accurately and reproducibly. The measured results using the optical sensors were well in accordance with measurements using electrodes with minimal experimental variations. The sensors were further applied for evaluation of photosynthetic activities of Synechocystis sp. PCC 6803 at the exponential and stationary phases. The results were consistent with biological observation that the photosynthetic activity in the exponential phase was higher than that in the stationary phase.

A series of naphthalimide derivatives as intra and extracellular pH sensors.

A series of new naphthalimide derivatives were synthesized and studied. Three of the materials (SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and pK(a) values were studied. The film P3 composed of the sensing moiety SM3 has a pK(a) close to the usual biological environmental pH of approximately 7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli (E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their pK(a) values are in a range of 5.9-6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 mum in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of approximately 7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82-95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red under confocal fluorescence microscopy.

2,1,3-Benzothiadiazole (BTD)-moiety-containing red emitter conjugated amphiphilic poly(ethylene glycol)-block-poly(epsilon-caprolactone) copolymers for bioimaging.

2,1,3-Benzothiadiazole (BTD)-containing red emitter was chemically conjugated onto amphiphilic poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PEG-b-PCL) copolymers to form two new fluorophore-conjugated block copolymers (P5 and P7). P5 is a cationic amino group-containing polymer, whereas, P7 is a neutral polymer. The polymers formed micelles in aqueous solution with average diameters of 45 nm (P7) and 78 nm (P5), which were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM). Cell internalization of the micelles using mouse macrophage RAW 264.7 was investigated. The micelles formed from P5 were endocytosed into the cell's cytoplasm through a non-specific endocytosis process, which was affected by temperature and calcium ions. Micelles formed from P7 could not be endocytosed. The dramatic difference of cell uptake between P5 and P7 indicated the cationic amino groups had a strong influence on the cell internalization to enhance the endocytosis pathway. 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay was used to evaluate the cytotoxicity of the P5 micelle and no significant toxicity was observed. This study is the first report regarding the synthesis of BTD-conjugated block copolymers and the application of the biomacromolecules for bioimaging.