Redox activity and diffusion of hydrophilic, hydrophobic, and amphiphilic redox active molecules in a bicontinuous cubic phase

The objective was to examine how a bicontinuous cubic phase influences the diffusion and electrochemical activity of dissolved molecules. The cubic phase is a structure with three-dimensional continuous channels of water separated by an apolar membrane. A redox active molecule can dissolve in three different environments. A hydrophobic molecule will prefer the interior of the membrane, a hydrophilic molecule will prefer the water channels, and an amphiphilic molecule will be situated with its headgroup at the surface of the membrane and its tail in the interior. The electrochemical activity was measured with cyclic voltammetry and the transport behavior with chronocoulometry. All the molecules were redox active in the cubic phase; that is, all the molecules could reach the surface of the electrode and react. The cubic phase made the kinetics of the charge transfer slower, showing a quasi-reversible behavior. The reason may be that a layer of the membrane adheres to the hydrophobic electrode surface. The diffusion experiment showed that the diffusion was slower than in solution. The molecules that were restricted to diffuse within the membrane gave particularly low mass transport rates.     

Ultraviolet B radiation induces upregulation of calcitonin gene-related peptide levels in human Finn chamber skin samples

One of the neuropeptides that plays a role in UVB-induced immunomodulation is calcitonin gene-related peptide (CGRP), as demonstrated in several animal studies. CGRP can be detected in human skin, but effects of UVB exposure on CGRP levels in human skin are not known. We determined CGRP levels in human Finn chamber skin samples of 15 UVB-irradiated and 10 control volunteers. Filter samples were collected prior to and immediately after a UVB exposure protocol (5 consecutive days, with one personally determined minimal erythema dose (MED(jp)) per day). CGRP levels in filter samples were determined using a commercially available radioimmunoassay kit. CGRP could be detected in the filter samples and volunteers showed statistically significantly increased levels after UVB exposure. In addition, the CGRP levels of UVB-exposed volunteers were positively correlated with the dose of UVB in J/m(2) that they received on 5 consecutive days. In other words, higher UVB doses resulted in higher CGRP levels. In summary, CGRP, a mediator in UVB-induced immunomodulation, could be detected in human Finn chamber skin samples, and was significantly increased after UVB exposure. The CGRP level appeared to depend on the amount of UVB the volunteers received.     

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.     

Modified nucleosides in human serum.

Methylated purines and pyrimidines derived from the degradation of transfer ribonucleic acid have been shown to be excreted in abnormal amounts in the urine of patients with cancer. Recent technology developed by Gehrke and Kuo has allowed the separation and quantification of modified nucleosides in serum using reversed-phase high-performance liquid chromatography with diode-array measurement. Serum levels of ten modified nucleosides were measured in 37 normal healthy adults to establish normal values and to correlate activity with age and sex. In addition, serum levels of patients with several malignancies were measured to determine activity in these diseases. Levels of modified nucleosides in normal individuals were consistently reproducible and showed no significant variation among males versus females or with age. Patients with malignant diseases showed consistent elevations and these were highest in patients with more advanced disease. The evidence of no significant differences in the mean levels of modified nucleosides in serum with age or sex in normal adults and elevations in patients with malignancies demonstrate the potential value of modified nucleosides as cancer biomarkers.     

Effect of trace amounts of water on organic solvent transport through gamma-alumina membranes with varying pore sizes

The transport behavior of toluene and n-hexane in gamma-alumina membranes with different pore diameters was studied. It was shown that the permeability of water-lean hexane and toluene is in agreement with Darcy's law down to membrane pore diameters of 3.5 nm. The presence of molar water fractions of 5-8 x 10(-4) in these solvents led to a permeability decrease of the gamma-alumina layer by a factor of 2-4 depending on pore size. In general, a lower permeability was found for hexane than for toluene. Moreover, in the presence of water a minimum applied pressure of 0.5-1.5 bar was required to induce net liquid flow through the membrane. These phenomena were interpreted in terms of capillary condensation of water in membrane pores with a size below a certain critical diameter. This is thought to lead to substantial blocking of these pores for transport, so that the effective tortuosity of the membrane for transport of hydrophobic solvents increases.     

Synthesis and conformational analysis by 1H NMR and restrained molecular dynamics simulations of the cyclic decapeptide [Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly]

Summary The design of enzyme mimics with therapeutic and industrial applications has interested both experimental and computational chemists for several decades. Recent advances in the computational methodology of restrained molecular dynamics, used in conjunction with data obtained from two-dimensional ¹H NMR spectroscopy, make it a promising method to study peptide and protein structure and function. Several issues, however, need to be addressed in order to assess the validity of this method for its explanatory and predictive value. Among the issues addressed in this study are: the accuracy and generizability of the GROMOS peptide molecular mechanics force field; the effect of inclusion of solvent on the simulations; and the effect of different types of restraining algorithms on the computational results. The decapeptide Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, which corresponds to the sequence of ACTH_1–10, has been synthesized, cyclized, and studied by two-dimensional ¹H NMR spectroscopy. Restrained molecular dynamics (RMD) and time-averaged restrained molecular dynamics (TARMD) simulations were carried out on four different distance-geometry starting structures in order to determine and contrast the behavior of cyclic ACTH_1–10 in vacuum and in solution. For the RMD simulations, the structures did not fit the NOE data well, even at high values of the restraining potential. The TARMD simulation method, however, was able to give structures that fit the NOE data at high values of the restraining potential. In both cases, inclusion of explicit solvent molecules in the simulation had little effect on the quality of the fit, although it was found to dampen the motion of the cyclic peptide. For both simulation techniques, the number and size of the NOE violations increased as the restraining potential approached zero. This is due, presumably, to inadequacies in the force field. Additional TARMD vacuum-phase simulations, run with a larger memory length or with a larger sampling size (16 additional distance-geometry structures), yielded no significantly different results. The computed data were then analyzed to help explain the sparse NOE data and poor chymotryptic activity of the cyclic peptide. Cyclic ACTH_1–10, which contains the functional moieties of the catalytic triad of chymotrypsin, was evaluated as a potential mimic of chymotrypsin by measurement of the rate of hydrolysis of esters of L-and d-phenylalanine. The poor rate of hydrolysis is attributed to the flexibility of the decapeptide, the motion of the side chains, which result in the absence of long-range NOEs, the small size of the macrocycle relative to that of the substrate, and the inappropriate orientation of the Gly, His, and Ser residues. The results demonstrate the utility of this method in computer-aided molecular design of cyclic peptides and suggest structural modifications for future work based on a larger and more rigid peptide framework.     

PHOTOSENSITIZING PROPERTIES OF BACTERIOCHLOROPHYLLIN a and BACTERIOCHLORIN a, TWO DERIVATIVES OF BACTERIOCHLOROPHYLL a

Abstract The photosensitizing properties of two water soluble derivatives of bacteriochlorophyll a , bacteriochlorophyllin a and bacteriochlorin a (lacking the central Mg-ion) were investigated and compared to those of hematoporphyrin derivatives. At physiological pH the oxygen consumption rate of histidine, tryptophan, dithiothreitol and guanosine upon illumination was 3 to 4 times higher when bacteriochlorin a was used as photosensitizer than when hematoporphyrin derivatives were used. Especially bacteriochlorin a proved to be an effective sensitizer for the killing of L929 cells. Because bacteriochlorin a has an absorption maximum at 765 nm in phosphate buffered saline (allowing a light penetration in tissue about ten times larger than at 630 nm) and a high molar absorption coefficient (32 000 M cm⁻¹) it has promising possibilities for the application in photodynamic therapy.     

Selective pervaporation of water through a nonselective microporous titania membrane by a dynamically induced molecular sieving mechanism

Pervaporation experiments were performed on microporous titania membranes using several binary liquids containing 2-20 wt % water. The membrane was nonselective in the separation of water from alcohols and p-dioxane but showed a remarkably high selectivity in the separation of water from ethylene glycol/water mixtures with < or =15 mol % water. The absence of selectivity under most conditions is explained by the large pore size (0.9 nm) of microporous titania. The high selectivity for water in the separation from ethylene glycol can be explained by the formation of a hydrogen-bonded network of ethylene glycol in the micropores, which blocks transport of ethylene glycol, while water can still permeate through. These networks are disrupted by water at higher concentrations, leading to full loss of membrane selectivity.     

Molecular structure of ethynylbenzene from electron diffraction and ab initio molecular orbital calculations

The molecular structure and benzene ring distortions of ethynylbenzene have been investigated by gas-phase electron diffraction and ab initio MO calculations at the HF/6-31G^* and 6-3G^** levels. Least-squares refinement of a model withC _2v, symmetry, with constraints from the MO calculations, yielded the following important bond distances and angles:r _g(C _i -C _o )=1.407±0.003 Å,r _g(C _o -C _m )=1.397±0.003 Å,r _g(C _m -C _p )=1.400±0.003 Å,r _g(Cr _i -CCH)=1.436 ±0.004 Å,r _g(C=C)=1.205±0.005 Å, C _o -C _i -C _o =119.8±0.4°. The deformation of the benzene ring of ethynylbenzene given by the MO calculations, including o-C_i-C_o=119.4°, is insensitive to the basis set used and agrees with that obtained by low-temperature X-ray crystallography for the phenylethynyl fragment, C₆H₅-CC-, in two different crystal environments. The partial substitution structure of ethynylbenzene from microwave spectroscopy is shown to be inaccurate in the ipso region of the benzene ring.