Skeletal muscle (1) H MRSI before and after prolonged exercise. I. muscle specific depletion of intramyocellular lipids

Aim of the study was to determine distribution and depletion patterns of intramyocellular lipids (IMCL) in leg muscles before and after two types of standardized endurance exercise. ¹H-magnetic resonance spectroscopic imaging was performed (1) in the thigh of eight-trained cyclists after exercising on an ergometer for 3 h at 52 ± 8% of maximal speed and (2) in the lower leg of eight-trained runners after exercising on a treadmill for 3 h at 49 ± 3% of maximal workload. Pre-exercise IMCL contents were reduced postexercise in 11 out of 13 investigated upper and lower leg muscles (P < 0.015 for all). A strong linear correlation with a slope of ∼0.5 between pre-exercise IMCL content and IMCL depletion was found. IMCL depletion differed strongly between muscles. Absolute and also relative IMCL reduction was significantly higher in muscles with predominantly slow fibers compared to those with fast fibers. Creatine levels and fiber orientation were stable and unchanged after exercise, while trimethyl-ammonium groups increased. This is presented in the accompanying paper. In conclusion, a systematic comparison of metabolic changes in cross sections of the upper and lower leg was performed. The results imply that pre-exercise IMCL levels determine the degree of IMCL depletion after exercise.

Conventional and anti-erosion fluoride toothpastes: effect on enamel erosion and erosion-abrasion

New toothpastes with anti-erosion claims are marketed, but little is known about their effectiveness. This study investigates these products in comparison with various conventional NaF toothpastes and tin-containing products with respect to their erosion protection/abrasion prevention properties. In experiment 1, samples were demineralised (10 days, 6 × 2 min/day; citric acid, pH 2.4), exposed to toothpaste slurries (2 × 2 min/day) and intermittently stored in a mineral salt solution. In experiment 2, samples were additionally brushed for 15 s during the slurry immersion time. Study products were 8 conventional NaF toothpastes (1,400-1,490 ppm F), 4 formulations with anti-erosion claims (2 F toothpastes: NaF + KNO(3) and NaF + hydroxyapatite; and 2 F-free toothpastes: zinc-carbonate-hydroxyapatite, and chitosan) and 2 Sn-containing products (toothpaste: 3,436 ppm Sn, 1,450 ppm F as SnF(2)/NaF; gel: 970 ppm F, 3,030 ppm Sn as SnF(2)). A mouth rinse (500 ppm F as AmF/NaF, 800 ppm Sn as SnCl(2)) was the positive control. Tissue loss was quantified profilometrically. In experiment 1, most NaF toothpastes and 1 F-free formulation reduced tissue loss significantly (between 19 and 42%); the Sn-containing formulations were the most effective (toothpaste and gel 55 and 78% reduction, respectively). In experiment 2, only 4 NaF toothpastes revealed significant effects compared to the F-free control (reduction between 29 and 37%); the F-free special preparations and the Sn toothpaste had no significant effect. The Sn gel (reduction 75%) revealed the best result. Conventional NaF toothpastes reduced the erosive tissue loss, but had limited efficacy regarding the prevention of brushing abrasion. The special formulations were not superior, or were even less effective.

The plasma carnitine concentration regulates renal OCTN2 expression and carnitine transport in rats

Previous findings in rats and in human vegetarians suggest that the plasma carnitine concentration and/or carnitine ingestion may influence the renal reabsorption of carnitine. We tested this hypothesis in rats with secondary carnitine deficiency following treatment with N-trimethyl-hydrazine-3-propionate (THP) for 2 weeks and rats treated with excess L-carnitine for 2 weeks. Compared to untreated control rats, treatment with THP was associated with an approximately 70% decrease in plasma carnitine and with a 74% decrease in the skeletal muscle carnitine content. In contrast, treatment with L-carnitine increased plasma carnitine levels by 80% and the skeletal muscle carnitine content by 50%. Treatment with L-carnitine affected neither the activity of carnitine transport into isolated renal brush border membrane vesicles, nor renal mRNA expression of the carnitine transporter OCTN2. In contrast, in carnitine deficient rats, carnitine transport into isolated brush border membrane vesicles was increased 1.9-fold compared to untreated control rats. Similarly, renal mRNA expression of OCTN2 increased by a factor of 1.7 in carnitine deficient rats, whereas OCTN2 mRNA expression remained unchanged in gut, liver or skeletal muscle. Our study supports the hypothesis that a decrease in the carnitine plasma and/or glomerular filtrate concentration increases renal expression and activity of OCTN2.

gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention

gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol. gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol. gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine. gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance. Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.

Supported Cu(II) polymer catalysts for aqueous phenol oxidation

Supported Cu(II) polymer catalysts were used for the catalytic oxidation of phenol at 30 degrees C and atmospheric pressure using air and H(2)O(2) as oxidants. Heterogenisation of homogeneous Cu(II) catalysts was achieved by adsorption of Cu(II) salts onto polymeric matrices (poly(4-vinylpyridine), Chitosan). The catalytic active sites were represented by Cu(II) ions and showed to conserve their oxidative activity in heterogeneous catalysis as well as in homogeneous systems. The catalytic deactivation was evaluated by quantifying released Cu(II) ions in solution during oxidation, from where Cu-PVP(25) showed the best leaching levels no more than 5 mg L(-1). Results also indicated that Cu-PVP(25) had a catalytic activity (56% of phenol conversion when initial Cu(II) catalytic content was 200 mg L(Reaction)(-1)) comparable to that of commercial catalysts (59% of phenol conversion). Finally, the balance between activity and copper leaching was better represented by Cu-PVP(25) due to the heterogeneous catalytic activity had 86% performance in the heterogeneous phase, and the rest on the homogeneous phase, while Cu-PVP(2) had 59% and CuO/gamma-Al(2)O(3) 68%.

ALTERNATING CURRENT DIELECTROPHORESIS OF CORE-SHELL NANOPARTICLES: EXPERIMENTS AND COMPARISON WITH THEORY

Nanoparticles are fascinating where physical and optical properties are related to size. Highly controllable synthesis methods and nanoparticle assembly are essential [6] for highly innovative technological applications. Among nanoparticles, nonhomogeneous core-shell nanoparticles (CSnp) have new properties that arise when varying the relative dimensions of the core and the shell. This CSnp structure enables various optical resonances, and engineered energy barriers, in addition to the high charge to surface ratio. Assembly of homogeneous nanoparticles into functional structures has become ubiquitous in biosensors (i.e. optical labeling) [7, 8], nanocoatings [9-13], and electrical circuits [14, 15]. Limited nonhomogenous nanoparticle assembly has only been explored. Many conventional nanoparticle assembly methods exist, but this work explores dielectrophoresis (DEP) as a new method. DEP is particle polarization via non-uniform electric fields while suspended in conductive fluids. Most prior DEP efforts involve microscale particles. Prior work on core-shell nanoparticle assemblies and separately, nanoparticle characterizations with dielectrophoresis and electrorotation [2-5], did not systematically explore particle size, dielectric properties (permittivity and electrical conductivity), shell thickness, particle concentration, medium conductivity, and frequency. This work is the first, to the best of our knowledge, to systematically examine these dielectrophoretic properties for core-shell nanoparticles. Further, we conduct a parametric fitting to traditional core-shell models. These biocompatible core-shell nanoparticles were studied to fill a knowledge gap in the DEP field. Experimental results (chapter 5) first examine medium conductivity, size and shell material dependencies of dielectrophoretic behaviors of spherical CSnp into 2D and 3D particle-assemblies. Chitosan (amino sugar) and poly-L-lysine (amino acid, PLL) CSnp shell materials were custom synthesized around a hollow (gas) core by utilizing a phospholipid micelle around a volatile fluid templating for the shell material; this approach proves to be novel and distinct from conventional core-shell models wherein a conductive core is coated with an insulative shell. Experiments were conducted within a 100 nl chamber housing 100 um wide Ti/Au quadrapole electrodes spaced 25 um apart. Frequencies from 100kHz to 80MHz at fixed local field of 5Vpp were tested with 10-5 and 10-3 S/m medium conductivities for 25 seconds. Dielectrophoretic responses of ~220 and 340(or ~400) nm chitosan or PLL CSnp were compiled as a function of medium conductivity, size and shell material.

Virus purification, detection and removal

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus vaccine recovery can be as low as 30% due to demand of high final titer, virus transduction inhibitors and presence of empty or defective virus capsids as impurities. We have overcome these shortcomings by recovering a high 64% of infectious virus using an aqueous two-phase system. We used high molecular weight polymer and citrate salt to achieve a good yield and eliminated the major contaminant bovine serum albumin. Viruses are also studied for ensuring pure and safe drinking water. Low pressure microfiltrations are continuously being investigated for water filters as they allow high permeate flux and low fouling. Viruses such as PPV are small enough to pass through the microporous membranes. Control of viruses in water is crucial for public health and we have designed an affinity based membrane filter to capture virus. Nanofibers have a high surface to volume ratio providing a highly accessible surface area for virus adsorption. Chitosan an insoluble, biocompatible and biodegradable polymer was used for adsorbing trimer peptide WRW. About 0.2 μmoles of cysteine terminal WRW peptide was conjugated to amine terminal chitosan using maleimide conjugation chemistry. We achieved 90-99% virus removal from water adjusted to a neutral pH. The virus removal from affinity based chitosan was attributed to electrostatic and hydrophobic driven binding effect.

DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYMERIC MICELLES FOR GENE-DIRECTED ENZYME PRODRUG THERAPY

Gene-directed enzyme prodrug therapy is a form of cancer therapy in which delivery of a gene that encodes an enzyme is able to convert a prodrug, a pharmacologically inactive molecule, into a potent cytotoxin. Currently delivery of gene and prodrug is a two-step process. Here, we propose a one-step method using polymer nanocarriers to deliver prodrug, gene and cytotoxic drug simultaneously to malignant cells. Prodrugs acyclovir, ganciclovir and 5-doxifluridine were used to directly to initiate ring-opening polymerization of epsilon-caprolactone, forming a hydrophobic prodrug-tagged poly(epsilon-caprolactone) which was further grafted with hydrophilic polymers (methoxy poly(ethylene glycol), chitosan or polyethylenemine) to form amphiphilic copolymers for micelle formation. Successful synthesis of copolymers and micelle formation was confirmed by standard analytical means. Conversion of prodrugs to their cytotoxic forms was analyzed by both two-step and one-step means i.e. by first delivering gene plasmid into cell line HT29 and then challenging the cells with the prodrug-tagged micelle carriers and secondly by complexing gene plasmid onto micelle nanocarriers and delivery gene and prodrug simultaneously to parental HT29 cells. Anticancer effectiveness of prodrug-tagged micelles was further enhanced by encapsulating chemotherapy drugs doxorubicin or SN-38. Viability of colon cancer cell line HT29 was significantly reduced. Furthermore, in an effort to develop a stealth and targeted carrier, CD47-streptavidin fusion protein was attached onto the micelle surface utilizing biotin-streptavidin affinity. CD47, a marker of self on the red blood cell surface, was used for its antiphagocytic efficacy, results showed that micelles bound with CD47 showed antiphagocytic efficacy when exposed to J774A.1 macrophages. Since CD47 is not only an antiphagocytic ligand but also an integrin associated protein, it was used to target integrin alpha(v)beta(3), which is overexpressed on tumor-activated neovascular endothelial cells. Results showed that CD47-tagged micelles had enhanced uptake when treated to PC3 cells which have high expression of alpha(v)beta(3). The synthesized multifunctional polymeric micelle carriers developed could offer a new platform for an innovative cancer therapy regime.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-A density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.

Cardiac lipid levels show diurnal changes and long-term variations in healthy human subjects

(1) H-MRS is regularly applied to determine lipid content in ectopic tissue - mostly skeletal muscle and liver - to investigate physiological and/or pathologic conditions, e.g. insulin resistance. Technical developments also allow non-invasive in vivo assessment of cardiac lipids; however, basic data about methodological reliability (repeatability) and physiological variations are scarce. The aim of the presented work was to determine potential diurnal changes of cardiac lipid stores in humans, and to put the results in relation to methodological repeatability and normal physiological day-to-day variations. Optimized cardiac- and respiratory-gated (1) H-MRS was used for non-invasive quantification of intracardiomyocellular lipids (ICCL), creatine, trimethyl-ammonium compounds (TMA), and taurine in nine healthy young men at three time points per day on two days separated by one week. This design allowed determination of (a) diurnal changes, (b) physiological variation over one week and (c) methodological repeatability of the ICCL levels. Comparison of fasted morning to post-absorptive evening measurements revealed a significant 37 ± 19% decrease of ICCL during the day (p = 0.0001). There was a significant linear correlation between ICCL levels in the morning and their decrease during the day (p = 0.015). Methodological repeatability for the ICCL/creatine ratio was excellent, with a coefficient of variance of ~5%, whereas physiological variation was found to be considerably higher (22%) in spite of a standardized physiological preparation protocol. In contrast, TMA levels remained stable over this time period. The proposed (1) H-MRS technique provides a robust way to investigate relevant physiological changes in cardiac metabolites, in particular ICCL. The present results suggest that ICCL reveal a diurnal course, with higher levels in the morning as compared to evening. In addition, a considerable long-term variation of ICCL levels, in both the morning and evening, was documented. Given the high methodological repeatability, these effects should be taken into account in studies investigating the metabolic role of ICCL.

Benzoxazinoid Metabolites Regulate Innate Immunity against Aphids and Fungi in Maize

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

The Effects of Physical and Chemical Modulating Agents on the Induction of Vaccine-Induced Immune Responses

After the development of the viral-based prostate cancer vaccine, Ad5-PSA, much research has been orientated to help enhance the induced immune response by combining the vaccine with physical and chemical modulating agents, more specifically the polymers polyethylenimine (PEI), chitosan, and chitosan coated with CD3 complex antibodies; all previously shown to stimulate an immune response as isolated gene carriers. To compare the vaccine-induced immune responses between the naked vaccine and the polymer-vaccine combinations, a mouse model using the ovalbumin- specific Ad-OVA vaccine was tested using intracellular cytokine staining (ICS), tetramer staining, and cytotoxic T-cell lymphocyte assays to measure the activation of CD8+ T-cells, interferon gamma proteins (INFƒ×), and the induced cytotoxicity to ovalbumin. The Ad-OVA vaccine combined with both chitosan and chitosan with CD3 complex antibodies, both natural polymers, were found to induce similar immune responses to the naked vaccine while the vaccine combined with the synthetic polymer, PEI, diminished the immune response.

A comparison between multi-proxy and historical data of drift-ice conditions on the East Greenland shelf

The reduction in sea ice along the SE Greenland coast during the last century has severely impacted ice-rafting to this area. In order to reconstruct ice-rafting and oceanographic conditions in the area of Denmark Strait during the last ~150 years, we conducted a multiproxy study on three short (20 cm) sediment cores from outer Kangerdlugssuaq Trough (~300 m water depth). The proxy-based data obtained have been compared with historical and instrumental data to gain a better understanding of the ice sheet-ocean interactions in the area. A robust chronology has been developed based on 210Pb and 137Cs measurements on core PO175GKC#9 (~66.2°N, 32°W) and expanded to the two adjacent cores based on correlations between calcite weight percent records. Our proxy records include sea-ice and phytoplankton biomarkers, and a variety of mineralogical determinations based on the <2 mm sediment fraction, including identification with quantitative x-ray diffraction, ice-rafted debris counts on the 63-150 µm sand fraction, and source identifications based on the composition of Fe oxides in the 45-250 µm fraction. A multivariate statistical analysis indicated significant correlations between our proxy records and historical data, especially with the mean annual temperature data from Stykkishólmur (Iceland) and the storis index (historical observations of sea-ice export via the East Greenland Current). In particular, the biological proxies (calcite weight percent, IP25, and total organic carbon %) showed significant linkage with the storis index. Our records show two distinct intervals in the recent history of the SE Greenland coast. The first of these (ad 1850-1910) shows predominantly perennial sea-ice conditions in the area, while the second (ad 1910-1990) shows more seasonally open water conditions.