Coffee consumption attenuates short-term fructose-induced liver insulin resistance in healthy men.

BACKGROUND Epidemiologic and experimental data have suggested that chlorogenic acid, which is a polyphenol contained in green coffee beans, prevents diet-induced hepatic steatosis and insulin resistance. OBJECTIVE We assessed whether the consumption of chlorogenic acid-rich coffee attenuates the effects of short-term fructose overfeeding, dietary conditions known to increase intrahepatocellular lipids (IHCLs), and blood triglyceride concentrations and to decrease hepatic insulin sensitivity in healthy humans. DESIGN Effects of 3 different coffees were assessed in 10 healthy volunteers in a randomized, controlled, crossover trial. IHCLs, hepatic glucose production (HGP) (by 6,6-d2 glucose dilution), and fasting lipid oxidation were measured after 14 d of consumption of caffeinated coffee high in chlorogenic acid (C-HCA), decaffeinated coffee high in chlorogenic acid, or decaffeinated coffee with regular amounts of chlorogenic acid (D-RCA); during the last 6 d of the study, the weight-maintenance diet of subjects was supplemented with 4 g fructose · kg(-1) · d(-1) (total energy intake ± SD: 143 ± 1% of weight-maintenance requirements). All participants were also studied without coffee supplementation, either with 4 g fructose · kg(-1) · d(-1) (high fructose only) or without high fructose (control). RESULTS Compared with the control diet, the high-fructose diet significantly increased IHCLs by 102 ± 36% and HGP by 16 ± 3% and decreased fasting lipid oxidation by 100 ± 29% (all P < 0.05). All 3 coffees significantly decreased HGP. Fasting lipid oxidation increased with C-HCA and D-RCA (P < 0.05). None of the 3 coffees significantly altered IHCLs. CONCLUSIONS Coffee consumption attenuates hepatic insulin resistance but not the increase of IHCLs induced by fructose overfeeding. This effect does not appear to be mediated by differences in the caffeine or chlorogenic acid content. This trial was registered at clinicaltrials.gov as NCT00827450.

Osseointegration of titanium implants functionalised with phosphoserine-tethered poly(epsilon-lysine) dendrons: a comparative study with traditional surface treatments in sheep

The aim of this study was to analyse the osseointegrative potential of phosphoserine-tethered dendrons when applied as surface functionalisation molecules on titanium implants in a sheep model after 2 and 8 weeks of implantation. Uncoated and dendron-coated implants were implanted in six sheep. Sandblasted and etched (SE) or porous additive manufactured (AM) implants with and without additional dendron functionalisation (SE-PSD; AM-PSD) were placed in the pelvic bone. Three implants per group were examined histologically and six implants were tested biomechanically. After 2 and 8 weeks the bone-to-implant contact (BIC) total values of SE implants (43.7 ± 12.2; 53.3 ± 9.0 %) and SE-PSD (46.7 ± 4.5; 61.7 ± 4.9 %) as well as AM implants (20.49 ± 5.1; 43.9 ± 9.7 %) and AM-PSD implants (19.7 ± 3.5; 48.3 ± 15.6 %) showed no statistically significant differences. For SE-PSD and AM-PSD a separate analysis of only the cancellous BIC demonstrated a statistically significant difference after 2 and 8 weeks. Biomechanical findings proved the overall increased stability of the porous implants after 8 weeks. Overall, the great effect of implant macro design on osseointegration was further supported by additional phosphoserine-tethered dendrons for SE and AM implants.

Effects of increased CO2 on fish gill and plasma proteome

Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen beta chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1 gamma, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.

Branched co-polymers of histidine and lysine are efficient carriers of plasmids

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.

Expression of the fructose transporter GLUT5 in human breast cancer.

The primary metabolic characteristic of malignant cells is an increased uptake of glucose and its anaerobic metabolism. We studied the expression and function of the glucose transporters in human breast cancer cell lines and analyzed their expression in normal and neoplastic primary human breast tissue. Hexose uptake assays and immunoblotting experiments revealed that the breast carcinoma cell lines MCF-7 and MDA-468 express the glucose transporters GLUT1 and GLUT2, isoforms expressed in both normal and neoplastic breast tissue. We also found that the breast cancer cell lines transport fructose and express the fructose transporter GLUT5. Immunolocalization studies revealed that GLUT5 is highly expressed in vivo in human breast cancer but is absent in normal human breast tissue. These findings indicate that human breast cancer cells have a specialized capacity to transport fructose, a metabolic substrate believed to be used by few human tissues. Identification of a high-affinity fructose transporter on human breast cancer cells opens opportunities to develop novel strategies for early diagnosis and treatment of breast cancer.

A defective signal peptide in the maize high-lysine mutant floury 2.

The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa alpha-zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa alpha-zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa alpha-zein protein with other alpha-zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this alpha-zein explain many of the phenotypic effects of the fl2 mutation.

La tagatose-1,6-bisphosphate aldolase et la fructose-1,6-bisphosphate aldolase de classe I : mécanisme et stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase qui fait preuve d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-bisphosphate (TBP), mais également du fructose-1,6-bisphosphate (FBP), du sorbose-1,6-bisphosphate et du psicose-1,6-bisphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Aldolase de classe I, qui donc catalyse sa réaction en formant un intermédiaire covalent obligatoire, ou base de Schiff, avec son susbtrat, la TBP aldolase de S. pyogenes partage 14 % d’identité avec l’enzyme modèle de cette famille, la FBP aldolase de muscle de mammifère. Bien que le mécanime catalytique de la FBP aldolase des mammifères ait été examiné en détails et qu’il soit approprié d’en tirer des renseignements quant à celui de la TBP aldolase, le manque singulier de stéréospécificité de cette dernière tant dans le sens du clivage que celui de la condensation n’est toujours pas éclairci. Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution des structures de l’enzyme native et mutée, en complexe avec des subtrats et des inhibiteurs compétitifs, à des résolutions comprises entre 1.8 Å et 2.5 Å. Le trempage des cristaux de TBP aldolase native et mutante dans une solution saturante de FBP ou TBP a en outre permis de piéger un authentique intermédiaire covalent lié à la Lys205, la lysine catalytique. La determination des profils pH de la TBP aldolase native et mutée, entreprise afin d'évaluer l’influence du pH sur la réaction de clivage du FBP et TBP et ìdentifier le(s) résidu(s) impliqué(s), en conjonction avec les données structurales apportées par la cristallographie, ont permis d’identifier sans équivoque Glu163 comme résidu responsable du clivage. En effet, le mode de liaison sensiblement différent des ligands utilisés selon la stéréochimie en leur C3 et C4 permet à Glu163, équivalent à Glu187 dans la FBP aldolase de classe I, d’abstraire le proton sur l’hydroxyle du C4 et ainsi d’amorcer le clivage du lien C3-C4. L’étude du mécanimse inverse, celui de la condensation, grâce par exemple à la structure de l’enzyme native en complexe avec ses substrats à trois carbones le DHAP et le G3P, a en outre permis d’identifier un isomérisme du substrat G3P comme possible cause de la synthèse des isomères en C4 par cette enzyme. Ce résultat, ainsi que la decouverte d’un possible isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP, identifié précedemment, permet de cerner presque complètement les particularités du mécanisme de cette enzyme et d’expliquer comment elle est capable de synthétiser les quatres stéréoisomères 3(S/R), 4(S/R). De plus, la résolution de ces structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. Enfin, la résolution de la structure du mutant Lys229→Met de la FBP aldolase de muscle en complexe avec la forme cyclique du FBP, de même que des études cristallographiques sur le mutant équivalent Lys205→Met de la TBP aldolase de S. pyogenes et des expériences de calorimétrie ont permis d’identifier deux résidus particuliers, Ala31 et Asp33 chez la FBP aldolase, comme possible cause de la discrimination de cette enzyme contre les substrats 3(R) et 4(S), et ce par encombrement stérique des substrats cycliques. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

The Effect of Fructose vs. Glucose Beverages on Low-Grade Systemic Inflammation

Thesis (Ph.D.)--University of Washington, 2016-04

Effects of phytase supplementation of phosphorus-adequate, lysine-deficient, wheat-based diets on growth performance of weaner pigs

The effects of microbial phytase supplementation of phosphorus-adequate, wheat-based diets with available lysine : energy density ratios ranging from 0.75 to 0.90 g available lysine/MJ DE on growth performance of weaner pigs were investigated in 3 studies. In the first study, increasing levels of dietary phytate depressed growth rates (P<0.08) and efficiency of feed conversion (P<0.01) and phytase supplementation enhanced growth rates (P<0.05) and tended to improve feed efficiency (P<0.15). There were no significant interactions between dietary phytate and phytase inclusion to support the hypothesis that dietary substrate levels of phytate govern responses to phytase. However, in this and other studies, percentage increases in efficiency of feed conversion generated by phytase were positively correlated to dietary phytate concentrations to a significant extent (P<0.005), so it is possible that dietary substrate levels are of importance to the magnitude of responses following phytase supplementation. Diets with 3 levels of protein, expressed as 0.80, 0.85, and 0.90 g available lysine/MJ DE, were offered to pigs without and with phytase in the second study. Protein/amino acid levels or lysine : energy density ratios did not influence growth performance, which was not expected. However, phytase tended to increase growth rates (P<0.08) and improved feed efficiency (P<0.01). Although it is believed that phytase may have a positive influence on protein utilisation, this was not demonstrated in this experiment. In the third study, the simultaneous inclusion of phytase and xylanase feed enzymes in wheat-based weaner diets did not increase growth performance responses in comparison with phytase alone. Individually, phytase improved feed efficiency (P<0.05) and numerically increased growth rates (P<0.25). Although responses in growth performance of weaner pigs following phytase supplementation lacked consistency, they were generally positive and indicative of anti-nutritive properties of phytate that are unrelated to P availability. That these positive responses were observed in diets with suboptimal available lysine : energy density ratios is consistent with the possibility that phytate has a negative influence on protein utilisation, which is ameliorated by phytase. However, these antinutritive effects and their underlying mechanisms need to be better defined if full advantage of the potential protein-sparing effects of microbial phytase is to be taken.

Glass transition behaviour of fructose

The glass transition temperature and the second transition (the endothermic change between the glass transition and melting temperatures) of fructose were studied. The thermal history strongly affected both transitions of fructose. Storage for 10 days at 22degreesC increased the dynamic glass transition temperature from 16 to 25degreesC and decreased the second transition of fructose from 110 to 98degreesC in the first differential scanning calorimetric (DSC) scan. The amplitude of the second transition increased slightly with storage time and reached 260% of the first transition for vacuum oven dried samples. The effect of thermal history on the glass transition temperature of fructose can be removed by scanning the sample in a DSC to 130degreesC. The effects of water content, glucose and sucrose on the two transitions were also investigated.

Increased adjuvant activity of minimal CD8 T cell peptides incorporated into lipid-core-peptides

A problem facing the use of subunit peptide and protein vaccines is their inability to stimulate protective immune responses. Many different approaches have been utilized to overcome this inefficient immune activation. The approach we have taken is to modify the vaccine antigen so that it now has adjuvant properties. To do this, multiple copies of minimal CD8 T cell epitopes were attached to a poly lysine lipid core. These constructs are known as lipid-core-peptides (LCP). The research presented here examines the adjuvant activity of LCP. Using mouse models, we were able to show that LCP were indeed able to activate antigen-presenting cells in vitro and to activate cytotoxic T-cell responses in vivo. More importantly, LCP were able to stimulate the development of a protective antitumour immune response.

The influence of dietary sodium chloride, Arginine : Lysine ratio, and methionine source on apparent ileal digestibility of arginine and lysine in acutely heat-stressed broilers

The present study was carried out to determine the ileal digestibility of Arg and Lys in acutely heatstressed broilers using diets varying in Arg:Lys ratio, NaCl concentration, and Met Source. Male broilers were maintained at 22degreesC from 21 to 33 d of age and then at 32degreesC from 33 to 38 d of age. From 28 to 38 d of age, birds were fed a diet with an Arg:Lys ratio of 1.05 and 3 g of supplemental NaCl/kg of diet with or without L-arg free base to increase the Arg:Lys to 1.35, and with or without 3 g/kg of additional NaCl. Methionine was supplied as equimolar amounts of DL-Met or 2-hydroxy-4-(methylthio)-butanoic acid in a 2 x 2 x 2 design. At 38 d of age, digesta were collected from the terminal ileum, and amino acid analyses were conducted on feed and digesta samples and compared with acid-insoluble ash (dietary celite) to calculate the apparent ileal digestibilities of Lys and Arg. Increasing the NaCl concentration and the presence of HMB significantly decreased the digestibility of both Arg and Lys, whereas increasing the Arg:Lys ratio increased the digestibility of only Arg but did increase BW gain (P = 0.08). An interaction between dietary NaCl and Arg:Lys ratio as well as the 3-way interaction suggested that dietary NaCl could affect the apparent ileal digestibility of Arg and Lys at certain Arg:Lys ratios and the response may be influenced by the Met source.

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

The production of fructose from carbohydrate feedstocks

A review of the general chromatographic theory and of continuous chromatographic techniques has been carried out. Three methods of inversion of sucrose to glucose and fructose in beet molasses were explored. These methods were the inversion of sucrose using the enzyme invertase, by the use of hydrochloric acid and the use of the resin Amberlite IR118 in the H+ form. The preferred method on economic and purity considerations was by the use of the enzyme invertase. The continuous chromatographic separation of inverted beet molasses resulting in a fructose rich product and a product containing glucose and other non-sugars was carried out using a semi-continuous counter-current chromatographic refiner (SCCR6), consisting of ten 10.8cm x 75cm long stainless steel columns packed with a calcium charged 8% cross-linked polystyrene resin Zerolit SRC 14. Based on the literature this is the first time such a continuous separation has been attempted. It was found that the cations present in beet molasses displaced the calcium ions from the resin resulting in poor separation of the glucose and fructose. Three methods of maintaining the calcium form of the resin during the continuous operation of the equipment were established. Passing a solution of calcium nitrate through the purge column for half a switch period was found to be most effective as there was no contamination of the main fructose rich product and the product concentrations were increased by 50%. When a 53% total solids (53 Brix) molasses feedstock was used, the throughput was 34.13kg sugar solids per m3 of resin per hour. Product purities of 97% fructose in fructose rich (FRP) and 96% glucose in the glucose rich (GRP) products were obtained with product concentrations of 10.93 %w/w for the FRP and 10.07 %w/w for the GRP. The effects of flowrates, temperature and background sugar concentration on the distribution coefficients of fructose, glucose, betaine and an ionic component of beet molasses were evaluated and general relationships derived. The computer simulation of inverted beet molasses separations on an SCCR system has been carried out successfully.

Biosynthesis and separation of dextran fructose mixtures in a chromatographic reactor

A literature review of work carried out on batch and continuous chromatographic biochemical reactor-separators has been made. The major part of this work has involved the development of a batch chromatographic reactor-separator for the production of dextran and fructose by the enzymatic action of the enzyme dextransucrase on sucrose. In this reactor, simultaneous reaction and separation occurs thus reducing downstream processing and isolation of products as compared to the existing industrial process. The chromatographic reactor consisted of a glass column packed with a stationary phase consisting of cross linked polysytrene resin in the calcium form. The mobile phase consisted of diluted dextransucrase in deionised water. Initial experiments were carried out on a reactor separtor which had an internal diameter of 0.97cm and length of 1.5m. To study the effect of scale up the reactor diameter was doubled to 1.94cm and length increased to 1.75m. The results have shown that the chromatographic reactor uses more enzyme than a conventional batch reactor for a given conversion of sucrose and that an increase in void volume results in higher conversions of sucrose. A comparison of the molecular weight distribution of dextran produced by the chromatographic reactor was made with that from a conventional batch reactor. The results have shown that the chromatographic reactor produces 30% more dextran of molecular weight greater than 150,000 daltons at 20% w/v sucrose concentration than conventional reactors. This is because some of the fructose molecules are prevented as acting as acceptors in the chromatographic reactor due to their removal from the reaction zone. In the conventional reactor this is not possible and therefore a greater proportion of low molecular weight dextran is produced which does not have much clinical use. A theoretical model was developed to describe the behaviour of the reactor separator and this model was simulated using a computer. The simulation predictions showed good agreement with experimental results at high eluent flowrates and low conversions.