Triplet state spectra and dynamics of geometric isomers of carotenoids

The observation of preferential binding of cis-carotenoids in purple bacterial photosynthetic reaction centers versus trans-isomers in antenna pigment protein complexes has led to the hypothesis that the natural selection of stereoisomers has physiological significance. In order to test this hypothesis, we have undertaken a systematic series of investigations comparing the optical spectroscopic properties and excited state dynamics of cis and trans isomers of carotenoids. The present work compares the triplet state spectra, lifetimes, and energy transfer rates of all-trans-spheroidene and 13,14-locked-cis-spheroidene, the latter of which is incapable of isomerizing to the all-trans configuration, and therefore provides a unique opportunity to examine the triplet state properties of a structurally stable cis molecule. The data reveal only small differences in spectra, decay dynamics, and transfer times and suggest there is little intrinsic advantage in either triplet energy transfer or triplet state decay arising from the inherently different isomeric forms of cis compared to trans carotenoids.     

Covalent linkages between cellulose and lignin in cell walls of coniferous and nonconiferous woods

Covalent linkages between wall polysaccharides and lignin, especially linkage between cellulose and lignin were discussed by carboxymethylation technique of whole cell walls of coniferous and nonconiferous woods. Hydroxyl groups of plant cell walls polysaccharides were highly substituted, but not those of lignin by carboxymethyl groups under the used conditions, and separated into water-soluble and insoluble fractions by water extraction. Carboxymethylated wall polysaccharides linked covalently with lignin were distributed into the water-insoluble fractions. Composition of carboxymethylated sugar residues in the both fractions was analyzed quantitatively by 1H NMR spectroscopy after hydrolyzation with D2SO4 in D2O. More than half of cellulose linked covalently with lignin in coniferous wood, but only one-sixth of cellulose was involved in the linkage in nonconiferous wood. The major noncellulosic wall polysaccharides of coniferous wood also linked significantly with lignin. On the other hand, noncellulosic wall polysaccharides of nonconiferous wood were involved slightly in the covalent linkage with lignin. The situation of linkage between wall polysaccharides containing cellulose and lignin was visualized by scanning electron micrographs.     

Continuous enzymatic transesterification of sesame oil and a fully hydrogenated fat: effects of reaction conditions on product characteristics

An immobilized lipase from Thermomyces lanuginosus (TL IM) was employed to mediate the continuous transesterification of sesame oil and fully hydrogenated soybean oil (FHSBO) in a packed-bed reactor operating at 70 degrees C. Reactions between sesame oil (rich in LLL (15.97%), LOL (31.56%), and OLO (21.15%) [L = linoleic; O = oleic]) and the fully hydrogenated fat ((73.7% SSS, 26.3% SPS) [S = stearic; P = palmitic]) produced semi-solid fats. These products are complex mixtures of triacylglycerol (TAG) species whose compositions depend on reaction conditions. The dependence of the steady state product TAG profile on space time was determined for four initial weight ratios of sesame oil to hydrogenated fat (90:10, 80:20, 70:30, and 60:40). Except for the trial involving a weight ratio of sesame oil to FHSBO of 60:40, near equilibrium conditions were achieved at space times of 30 min-1 h. The chemical, physical, and functional properties of the product semi-solid fats were characterized. The predominant TAG species in the quasi-equilibrium products obtained from the mixture initially containing 90% (w/w) sesame oil and 10% FHSBO were LOL (26.22%) and OLO (21.92%). For transesterification of 80% sesame oil and 20% FHSBO, the major product species were OOP (21.27%), LOL (17.46%), and OLO (13.93%). OOP (24.38%) was the major product for reaction of 70% sesame oil with 30% FHSBO. Appropriate choices of reaction conditions and initial ratios of sesame oil to FHSBO lead to TAG with melting profiles and solid fat contents (SFC) similar to those of a variety of commercial products.     

Separation and identification of neisserial lipooligosaccharide oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

We determined the optimal conditions for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS) released from neisserial lipooligosaccharides (LOS) by mild acid hydrolysis. We efficiently obtained detailed composition, sequence, and linkage information about high Mr LOS. We found that HPAE-PAD can discriminate isobaric (same Mr) molecules of different structure, for example, nLc4 and Gb4, distinguish alpha from beta chain extensions, and determine the number of phosphoethanolamine (PEA) substituents. HPAE-PAD provided quantitative information that could be used to compare the relative abundances of OS. We used HPAE-PAD to identify all of the known LOS alpha chain antennae. When used with antibody-binding profiles and exoglycosidase digestion results, HPAE-PAD can provide nearly complete structures rapidly.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.     

NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.     

A label-free continuous total-internal-reflection-fluorescence-based immunosensor

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (K(d)>10(-6)M), fast kinetics (k(off)>1s(-1)), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1-5 microM for panose, and 10-15 microM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated an enthalpic driven antigen-antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.     

Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Effects of environmental hypercapnia on animal physiology: a 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

Global climate change is associated with a progressive rise in ocean CO(2) concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO(2) stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of (13)C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10-15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO(2) concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.     

Diversity of Pseudomonas strains isolated with King's B and Gould's S1 agar determined by repetitive extragenic palindromic-polymerase chain reaction, 16S rDNA sequencing and Fourier transform infrared spectroscopy characterisation

King's B and Gould's S1 agar were compared with regard to the isolation of Pseudomonas from four environmental samples. In all samples, King's B gave the highest number of colony-forming units, and in some environments, there were more fluorescent colony-forming units on King's B as well. However, almost all types grew on Gould's S1, which enabled us to isolate a greater variety of groups than with King's B, fluorescent as well as non-fluorescent members of Pseudomonas. The Pseudomonas isolates were comparatively typed by repetitive extragenic palindromic-polymerase chain reaction and Fourier transform infrared spectroscopy, not previously used for environmental Pseudomonas. The two typing methods were similar in resolution, thus Fourier transform infrared spectroscopy proved fast and reproducible and is a good method for discrimination at subspecies level. Representative strains were identified by partial 16S rDNA sequencing. Thus, we suggest Gould's S1 agar be used for isolation of Pseudomonas because the results are reproducible, specific and give the most diverse recovery and the least work.