Background signal suppression by opposite polarity subtraction for targeted DNP NMR spectroscopy on mixture samples.

A novel method for background signal suppression is introduced to improve the selectivity of dynamic nuclear polarization (DNP) NMR spectroscopy in the study of target molecules within complex mixtures. The method uses subtraction between positively and negatively enhanced DNP spectra, leading to an improved contrast factor, which is the ratio between the target and background signal intensities. The proposed approach was experimentally validated using a reverse-micelle system that confines the target molecules together with the polarizing agent, OX063 trityl. A substantial increase in the contrast factor was observed, and the contrast factor was optimized through careful selection of the DNP build-up time. A simulation study based on the experimental results provides insights into a strategy for choosing the appropriate DNP build-up time and the corresponding selectivity of the method. Further analysis revealed a broad applicability of the technique, encompassing studies from large biomolecules to surface-modified polymers, depending on the nuclear spin diffusion rate with a range of gyromagnetic ratios.

Optimal-Control-Based Cß Chemical Shift Encoding for Efficient Signal Assignment of Solid Proteins.

Fast magic-angle spinning (MAS) solid-state NMR spectroscopy is a powerful tool for gaining structural and dynamic information on solid proteins. To access such information site-specifically, the signal assignment process is unavoidable. In the assignment process, Cα and Cß chemical shifts are of paramount importance in identifying the type of amino acid residues. Conventionally, however, recording the Cß chemical shift of solid proteins with relatively short transverse relaxation time is often limited by the long delay required for the magnetization transfer to Cß spins and its evolution, that is, by the sensitivity drop. In this article, we propose a new method that encodes the Cß chemical shifts onto the intensities of the scalar-coupled Cα signals by combining an optimal control-based spin manipulation pulse and a spin-state filter. This reduces the total required transverse evolution to less than half of that for the previously proposed method, opening up the concept of the Cß-encoding nearest-neighbor NMR, for the first time, to solid proteins. Also, the total measurement time was shorter than that required for the explicit Cß shift evolution. We demonstrate the sequential signal assignment for microcrystalline protein GB1, and then discuss the prospects for more challenging proteins.

Evolution of α-synuclein conformation ensemble toward amyloid fibril via liquid-liquid phase separation (LLPS) as investigated by dynamic nuclear polarization-enhanced solid-state MAS NMR.

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α-Synuclein (αSyn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of αSyn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13C-labeled His-50 of αSyn, we show that the distribution of the αSyn conformation is skewed from the initial completely random state to a loose ß-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose ß-rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets.

Nanodisc-to-Nanofiber Transition of Noncovalent Peptide-Phospholipid Assemblies.

We report a novel molecular architecture of peptide-phospholipid coassemblies. The amphiphilic peptide Ac-18A-NH2 (18A), which was designed to mimic apolipoprotein α-helices, has been shown to form nanodisc structures with phospholipid bilayers. We show that an 18A peptide cysteine substitution at residue 11, 18A[A11C], forms fibrous assemblies with 1-palmitoyl-2-oleoyl-phosphatidylcholine at a lipid-to-peptide (L/P) molar ratio of 1, a fiber diameter of 10-20 nm, and a length of more than 1 µm. Furthermore, 18A[A11C] can form nanodiscs with these lipid bilayers at L/P ratios of 4-6. The peptide adopts α-helical structures in both the nanodisc and nanofiber assemblies, although the α-helical bundle structures were evident only in the nanofibers, and the phospholipids of the nanofibers were not lamellar. Fluorescence spectroscopic analysis revealed that the peptide and lipid molecules in the nanofibers exhibited different solvent accessibility and hydrophobicity from those of the nanodiscs. Furthermore, the cysteine substitution at residue 11 did not result in disulfide bond formation, although it was responsible for the nanofiber formation, suggesting that this free sulfhydryl group has an important functional role. Alternatively, the disulfide dimer of 18A[A11C] preferentially formed nanodiscs, even at an L/P ratio of 1. Interconversions of these discoidal and fibrous assemblies were induced by the stepwise addition of free 18A[A11C] or liposomes into the solution. Furthermore, these structural transitions could also be induced by the introduction of oxidative and reductive stresses to the assemblies. Our results demonstrate that heteromolecular lipid-peptide complexes represent a novel approach to the construction of controllable and functional nanoscale assemblies.

Physico-chemical properties, wax composition, aroma profiles, and antioxidant activity of granulated non-centrifugal sugars from sugarcane cultivars of Thailand.

Non-centrifugal cane sugar (NCS) is globally consumed and has various health benefits. It is mostly produced in hardened block form, which is less convenient than in granulated form for food applications. In terms of the traditional processing of NCS, preparation of granulated products is difficult due to the impurities found in the cane juice extracted from the whole stalk. Therefore, the aim of this study was to characterize and determine the physico-chemical properties, wax composition (policosanols and long-chain aldehydes), volatile aroma profiles, and antioxidant activity of traditional NCS in granular form made from four different cane cultivars of Thailand. The total soluble solid, pH, color, and mineral content varied among the sugarcane cultivars, whereas there was no significant difference in the total sugar, phenolic and flavonoid content. The total policosanol, a cholesterol-lowering nutraceutical wax component, and long-chain aldehyde contents were similar in the NCS products amongst three cultivars, and ranged from 2.63 to 3.69 mg/100 g. The granulated NCS products, in which acetaldehyde and dimethyl sulfide were the main volatile compounds, gave less aroma components than traditional NCS. The use of different sugarcane cultivars thus influenced the quality attributes of granulated non-centrifugal sugar products.

Composition, Taste, Aroma, and Antioxidant Activity of Solidified Noncentrifugal Brown Sugars Prepared from Whole Stalk and Separated Pith of Sugarcane (Saccharum officinarum L.).

In this study, 2 types of solidified noncentrifugal brown sugars (W-NCS and P-NCS) were prepared from the whole stalk and separated pith, respectively, of raw sugarcane (Saccharum officinarum L.). These products were discriminated in terms of their quality attributes, including color, sugars and minerals composition, taste, aroma, and antioxidant activity. The brown color of P-NCS was clearly different compared with that of W-NCS with a color difference value (ΔE* ) of 9.36. There was no difference in the sugars and minerals composition between the 2 types of sugar, which led to very similar taste profiles. However, P-NCS had a weaker aroma intensity than W-NCS did. Moreover, P-NCS retained more than 60% of the antioxidant activity of W-NCS. The information gleaned from this study might be used to select appropriate end-uses for these 2 types of sugars.

Structure determination of uniformly (13)C, (15)N labeled protein using qualitative distance restraints from MAS solid-state (13)C-NMR observed paramagnetic relaxation enhancement.

Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn(2+) mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.

Changes in the physicochemical characteristics, including flavour components and Maillard reaction products, of non-centrifugal cane brown sugar during storage.

Changes in the quality attributes of non-centrifugal cane brown sugar represented by physicochemical characteristics as well as flavour components and Maillard reaction products (MRPs) were monitored every 3 months over 1 year of storage. Stored cane brown sugar became darker, and its moisture content and water activity (a(w)) increased during storage. Fructose and glucose levels decreased as non-enzymatic browning via the Maillard reaction occurred in the stored sample, and a similar trend was also discovered in aconitic and acetic acids. Stored cane brown sugar lost its acidic and sulfuric odours (58.70-39.35% and 1.85-0.08%, respectively); subsequently, the nutty and roasted aroma increased from 26.52% to 38.59% due to the volatile MRPs. The browning rate of stored cane brown sugar was positively associated with the development of volatile MRPs (Pearson's coefficient = 0.860), whereas the amount of 3-deoxyglucosone, an intermediate product of the Maillard reaction, had a lower association with the brown colour due to its relatively slow degradation rate.

Necrotizing fasciitis caused by Streptococcus pneumoniae.

A 69-year-old woman presented with shivering and pain in the lower extremities on 5 April 2006; she was referred to the dermatology division of our hospital on the following day with difficulty in walking. She had been suffering from non-viral, non-alcoholic liver cirrhosis, and was being treated by the Division of Internal Medicine. Physical examination showed edema in the lower extremities and light purpuras on the groin and legs. Low blood pressure had been observed since admission. Necrotizing fasciitis (NF) was suspected on the basis of the skin symptoms, systemic conditions, and magnetic resonance imaging. During surgical debridement under general anesthesia, cardiopulmonary arrest occurred, and the patient died 12 h after admission. NF, in its early stages, exhibits few skin changes. In order to differentiate it from other skin infections, it is necessary to take into account blood pressure, abnormal systemic conditions, and severe pain out of proportion to its minor skin changes. In the present case, Streptococcus pneumoniae was detected by blood culture. Soft tissue infectious diseases caused by S. pneumoniae, especially NF, are very rare. We have reviewed reported cases of NF caused by S. pneumoniae.

Antioxidant flavonoid glycosides from the leaves of Ficus pumila L.

The Okinawan folks in Japan use Ficus pumila L. as a beverage or herbal medicine to treat diabetes and high blood pressure. Four flavonoid glycosides were isolated and identified as rutin (1 and 3), apigenin 6-neohesperidose (2), kaempferol 3-robinobioside (4) and kaempferol 3-rutinoside (5). Among these compounds, rutin exhibited the strongest antioxidant activity in DPPH radical scavenging assay and superoxide radical inhibition assay. The preparation of Ooitabi leaves in water provide sufficient amount of flavonoid glycosides to the Okinawan although 50% of aqueous ethanol extracted these flavonoid glycosides more effectively. These results show the potential of Ooitabi leaves as a natural source of antioxidant for health management.

Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells.

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of (1)H NMR suggest that 2,4-di-O-sulfo-, 2-mono-O-sulfo- and 4-mono-O-sulfo-l-fucopyranose were involved in oversulfated fucoidan and 4-mono-O-sulfo-l-fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.

Inhibition of osteoporosis due to restricted food intake by the fish oils DHA and EPA and perilla oil in the rat.

Seven-week old female rats fed restricted foods including the fish oils Docosahesaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) and perilla oil with food intake decreased by 50%, had increases of fracture force and bone mineral density (BMD) and decreases in levels of Deoxypiridinoline (Dpd) and Calcium (Ca) in the urine, compared with those of rats with osteoporosis due to restricted soy bean oil food intake. Therefore, the fish oils DHA and EPA and perilla oil depressed excretion of urinary Ca and inhibited osteoporosis due to restricted food intake.

Running inhibits osteoporosis induced by protein-deficient (PD) food intake.

Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.

Inhibition of osteoporosis induced by protein deficient (PD) food intake by active vitamin D(3) and vitamin K(2) in rats.

Independent use of K(2) and D(3) and simultaneous application of K(2) and D(3) inhibited the development of osteoporosis caused by PD food intake. The ALP activity of urine as a marker of bone formation osteoporosis did not rise in rats fed PD foods containing D(3), K(2) or both together. Body and womb weights fell in rats fed PD foods with D(3) K(2) and both D(3), K(2). Osteoporosis caused by PD food intake found to be very similar to type II osteoporosis in respects of inhibition by D(3) and K(2) and rising urinary ALP activity.

Production of galactinol from sucrose by plant enzymes.

Galactinol, 1-O-(alpha-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material. UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots. Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source. Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves. The product was identified as galactinol by the retention times of HPLC, alpha-galactosidase digestion, and NMR spectrometry.

Inhibition of osteoporosis in rats fed with sugar cane wax.

Rats fed on a restricted, semi-purified diet containing a 50%-reduced level of carbohydrate and oil, but normal levels of protein, minerals and vitamins, exhibited osteoporosis. However, rats fed on this restricted diet, but containing sugar cane wax, did not exhibit this bone disease. Sugar cane wax, containing a long-chain carbohydrate with an OH radical, prevented the development of osteoporosis via a non-estrogenic mechanism.