Glycopeptide and Oligosaccharide Libraries

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.     

A digital microfluidic method for multiplexed cell-based apoptosis assays

Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.     

Enzymatic Oxygen Microsensor Based on Bilirubin Oxidase Applied to Microbial Fuel Cells Analysis

A selective oxygen biosensor based on bilirubin oxidase (BOx) was developed. The sensor was used for determining oxygen profiles in a membraneless, single‐chamber microbial fuel cell (SCMFC), fed with raw wastewater. The linear response of the sensor was optimized by a diffusion layer of silica gel. A computer‐controlled stage was used to obtain accurate and precise measurements. Oxygen concentration in biofilms covering electrodes was measured, showing 3 mg L^?1 of O₂ in the bulk solution, decreasing to 0 mg L^?1 in the cathodic biofilm. The MFC generated power in the range of 0–0.08 mW, associated to the oxygen content.     

Large Molecular Quadratic Hyperpolarizabilities in Donor/Acceptor-Substituted trans-Tetraammineruthenium(II) Complexes

A series of new Ru(II) complex salts trans-[Ru(NH(3))(4)(L(1))(L(2))](PF(6))(n) [n = 2, L(1) = 4-acetylpyridine (4-acpy) and L(2) = 4-(dimethylamino)pyridine (dmap) (1), 4-(dimethylamino)benzonitrile (dmabn) (2), 4-picoline (4-pic) (3), or 1-methylimidazole (1-MeIm) (4); n = 3, L(1) = N-methyl-4,4'-bipyridinium (MeQ(+)) and L(2) = dmap (6), dmabn (7), 1-MeIm (8), 4-acpy (9), or phenothiazine (PTZ) (10); n = 2, L(1) = dmap and L(2) = 4-pyridinecarboxaldehyde (pyca) (12) or ethyl isonicotinate (isne) (13)] have been synthesized and fully characterized. These complexes display intense, visible metal-to-ligand charge-transfer (MLCT) absorptions which are highly solvatochromic. An X-ray crystal structure determination has been carried out for trans-[Ru(NH(3))(4)(MeQ(+))(PTZ)](PF(6))(3).Me(2)CO (10.Me(2)CO). This salt, empirical formula C(26)H(38)F(18)N(7)OP(3)RuS, crystallizes in the hexagonal system, space group P6(3), with a = b = 17.853(4) ?, c = 21.514(6) ?, and Z = 6. The MeQ(+) ligand adopts an almost planar conformation, with a torsion angle of 9.6 degrees between the two pyridyl rings. The dipolar cations exhibit a strong projected component along the z axis, but crystal twinning precludes second-harmonic generation. Measurements of the first hyperpolarizability beta by using the hyper-Rayleigh scattering technique at 1064 nm yield very large values in the range (232-621) x 10(-30) esu, the largest being for trans-[Ru(NH(3))(4)(MeQ(+))(dmabn)](PF(6))(3) (7). These beta values are resonance enhanced via the MLCT excitations. A correlation between beta and the MLCT absorption energy confirms that this excitation is the primary contributor to beta. The two-level model yields static hyperpolarizabilities beta(0) in the range (10-130) x 10(-30) esu, with trans-[Ru(NH(3))(4)(MeQ(+))(dmap)](PF(6))(3) (6) having the largest. The beta(0) values of the complexes of the bipyridyl ligand MeQ(+) are larger than those of their analogues containing monopyridyl ligands because of extended conjugation. beta(0) correlates with the MLCT energy only when the MLCT absorption is sufficiently far from the second harmonic at 532 nm.     

Pyoluteorin derivatives. II. Synthetic approaches to pyrrole ring substituted pyoluteorins

A method for the regiospecific synthesis of 3-substituted 2-aroylpyrroles is described. These pyrroles, which are structurally related to the naturally occurring antibiotic pyoluteorin, are prepared by a Friedel-Crafts aroylation of 4-substituted pyrrole-3-carboxylic acid esters with 2,6-dimethoxybenzoyl chloride. The carboalkoxy group is then removed by hydrolysis and decarboxylation to produce isomerically pure 3-substituted-2-(2′,6′-dimethoxybenzoyl)pyrroles ( 5 and 13 ). Conversion of these pyrroles into pyoluteorin-like compounds led to some unexpected products which arise from facile cleavage of the dihydroxybenzoyl portion of the molecules during chlorination.     

Synthesis and sensing of bisphosphonophosphate alkyl monoesters: A novel class of compounds for the study of nucleoside 5'-triphosphate chemistry

A series of novel β,γ-methylene-, monofluoromethylene-, and difluoromethylene-bisphosphonophosphate alkyl monoesters was synthesized. The compounds were conveniently detected during preparative HPLC using post-column derivatization with a phosphate-specific chemosensor.     

Enhanced effect of microdystrophin gene transfection by HSV-VP22 mediated intercellular protein transport

Background

Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo.

Results

Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice.

Conclusion

These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD.     

Structural,infra-red and optical measurements in some IV-VI alloy semiconductors

Single crystal alloy specimens of (GeSe)_x(SnSe)_1-x and (GeS)_x(SnS)_1-x have been prepared. The solubility limits, absorption edges, temperature coefficients of the energy gap and far infra-red reflectivities are investigated for the first time in these materials.     

Terahertz injection electroluminescence in multiperiod quantum-cascade AlGaAs/GaAs structures

Terahertz electroluminescence in the range ≈1.5 THz was observed in a quantum-cascade GaAs/AlGaAs structure containing 40 periods of tunnel-coupled wells. The luminescence is caused by the spatially indirect optical electron transitions between the ground states of neighboring quantum wells.