Exploring the Sulfatase 1 Catch Bond Free Energy Landscape using Jarzynski's Equality.

In non-covalent biological adhesion, molecular bonds commonly exhibit a monotonously decreasing life time when subjected to tensile forces (slip bonds). In contrast, catch bonds behave counter intuitively, as they show an increased life time within a certain force interval. To date only a hand full of catch bond displaying systems have been identified. In order to unveil their nature, a number of structural and phenomenological models have been introduced. Regardless of the individual causes for catch bond behavior, it appears evident that the free energy landscapes of these interactions bear more than one binding state. Here, we investigated the catch bond interaction between the hydrophilic domain of the human cell surface sulfatase 1 (Sulf1HD) and its physiological substrate heparan sulfate (HS) by atomic force microscopy based single molecule force spectroscopy (AFM-SMFS). Using Jarzynski's equality, we estimated the associated Gibbs free energy and provide a comprehensive thermodynamic and kinetic characterization of Sulf1HD/HS interaction. Interestingly, the binding potential landscape exhibits two distinct potential wells which confirms the recently suggested two state binding. Even though structural data of Sulf1HD is lacking, our results allow to draft a detailed picture of the directed and processive desulfation of HS.

A Threshold-Minimization Scheme for Exploring the Energy Landscape of Biomolecules: Application to a Cyclic Peptide and a Disaccharide.

We present a scheme, called the threshold-minimization method, for globally exploring the energy landscapes of small systems of biomolecular interest where typical exploration moves always require a certain degree of subsequent structural relaxation in order to be efficient, e.g., systems containing small or large circular carbon chains such as cyclic peptides or carbohydrates. We show that using this threshold-minimization method we can not only reproduce the global minimum and relevant local minima but also overcome energetic barriers associated with different types of isomerism for the example of a cyclic peptide, cyclo-(Gly)4. We then apply the new method to the disaccharide α-d-glucopyranose-1-2-ß-d-fructofuranose, report energetically preferred configurations and barriers to boat-chair isomerization in the glucopyranosyl ring, and discuss the energy landscape.

Fermentable oligosaccharide, disaccharide, monosaccharide and polyol content of foods commonly consumed by ethnic minority groups in the United Kingdom.

Dietary restriction of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) is an effective management approach for functional bowel disorders; however, its application is limited by the paucity of food composition data available for ethnic minority groups. The aim was to identify and measure the FODMAP content of these commonly consumed foods. According to their perceived importance to clinical practise, the top 20 ranked foods underwent FODMAP analysis using validated analytical techniques (total fructans, Megazyme hexokinase (HK) assay; all others, high-performance liquid chromatography (HPLC) with evaporative light scattering detectors). Of the 20 foods analysed, five were identified as significant sources of at least one FODMAP. Fructans and galacto-oligosaccharides were the major FODMAPs in these foods, including channa dal (0.13 g/100 g; 0.36 g/100 g), fenugreek seeds (1.11 g/100 g; 1.27 g/100 g), guava (0.41 g/100 g; not detected), karela (not detected; 1.12 g/100 g) and tamarind (2.35 g/100 g; 0.02 g/100 g). Broadening the availability of FODMAP composition data will increase the cultural application of low FODMAP dietary advice.

Arachidonic acid production by Mortierella alpina using raw crop materials.

BACKGROUND: Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance. METHODS: Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined. RESULTS: Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%. CONCLUSIONS: Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides.

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and ß-D-glucopyranosides, such as salicin share a common binding site of hTAS2R16.

Using the angle-dependent resonances of molded plasmonic crystals to improve the sensitivities of biosensors.

This paper describes how angle-dependent resonances from molded plasmonic crystals can be used to improve real-time biosensing. First, an inexpensive and massively parallel approach to create single-use, two-dimensional metal nanopyramidal gratings was developed. Second, although constant in bulk dielectric environments, the sensitivities (resonance wavelength shift and resonance width) of plasmonic crystals to adsorbed molecular layers of varying thickness were found to depend on incident excitation angle. Third, protein binding at dilute concentrations of protein was carried out at an angle that optimized the signal to noise of our plasmonic sensing platform. This angle-dependent sensitivity, which is intrinsic to grating-based sensors, is a critical parameter that can assist in maximizing signal to noise.

Dynamic restacking of Escherichia coli P-pili.

P-pili of uropathogenic Escherichia coli mediate the attachment to epithelial cells in the human urinary tract and kidney and therefore play an important role in infection. A better understanding of this mechanism could help to prevent bacteria from spreading but also provides interesting insights into molecular mechanics for future nanotech applications. The helical rod design of P-pili provides an efficient design to withstand hydrodynamic shear forces. The adhesive PapG unit at the distal end of the P-pilus forms a specific bond with the glycolipid Galabiose. This bond has a potential width Deltax = 0.7 +/- 0.15 nm and a dissociation rate K (Off) = 8.0.10(-4) +/- 5.0.10(-4) s(-1). It withstands a force of approximately 49 pN under physiological conditions. Additionally, we analyzed the behavior of unstacking and restacking of the P-pilus with dynamic force spectroscopy at velocities between 200 and 7,000 nm/s. Up to a critical extension of 66% of the totally stretched P-pilus, un/re-stacking was found to be fully reversible at velocities up to 200 nm/s. If the P-pilus is stretched beyond this critical extension a characteristic hysteresis appears upon restacking. This hysteresis originates from a nucleation process comparable to a first-order phase transition in an undercooled liquid. Analysis of the measurement data suggests that 20 PapA monomers are involved in the formation of a nucleation kernel.

Assessment of acid production by various human oral micro-organisms when palatinose or leucrose is utilized.

One promising way of reducing caries is by using sucrose substitutes in food, e.g., palatinose or leucrose. Previous experiments addressing cariogenic potential of sucrose substitutes have focused mainly on Streptococcus mutans. However, given the many other micro-organisms in the oral cavity, this study compared the acid production of 100 bacterial strains representing 44 different species, by batch fermentation in a test tube containing, as a sole carbohydrate source, glucose, sucrose, palatinose, or leucrose. Selected strains were further analyzed in a fermenter. Additionally, 30 yeast strains were tested by an auxanographic sugar assimilation test. Only Lactobacillus spp., Stomatococcus mucilaginosus, Leuconostoc mesenteroides, and Weissella paramesenteroides, and some of the yeasts studied-i.e., Candida albicans, C. tropicalis, C. parapsilosis, and Saccharomyces cerevisiae-utilized leucrose and/or palatinose well. Strikingly, Stomatococcus mucilaginosus produced water-insoluble polysaccharides by fermentation of leucrose and palatinose. In the fermenter, the respective sucrose substitutes were not only cleaved but also utilized. Thus, extracellular cleavage by autochthonous micro-organisms may produce cariogenic cleavage products (glucose, fructose) that can be used by other well-characterized cariogenic bacteria found in the oral flora. Therefore, the anticariogenic potential of sucrose substitutes in food might be limited.

A retrospective assessment of the clinical value of jejunal disaccharidase analysis.

BACKGROUND: The measurement of jejunal disaccharidases is used by several gastroenterologists when investigating suspected small-bowel disease. The clinical value of this analysis is assessed. METHOD: The histology and disaccharidase results in 1585 jejunal biopsy specimens were reviewed retrospectively. RESULTS: Disaccharidase and histology results concurred in most cases (72%). However, disaccharidases were an insensitive indicator of small-bowel disease: low levels were found in only 65% of coeliac patients with villous atrophy, 15% of patients with giardiasis, and 6% of patients with villous atrophy associated with non-coeliac histology. Low disaccharidase levels were sometimes found in patients with normal histology (1.6%) and when biopsy specimens were unwittingly taken from non-jejunal sites (1.4%). Isolated low lactase activities were found in 3.2%. Usually this finding was not clinically relevant because patients had no symptoms of lactose intolerance (38%), had another diagnosis that responded to appropriate treatment (8%), or had no response to a low-lactose diet (14%). In 16 patients sucrase activities were markedly low, and this investigation proved central to the diagnosis of sucrase-alpha-dextrinase deficiency, which was subsequently confirmed in 9. CONCLUSION: Jejunal disaccharidases are clinically useful only in the diagnosis of sucrase-alpha-dextrinase deficiency. We recommend that their measurement be reserved for the investigation of patients suspected of having this condition.

Nutritional assessment in humans and rats of leucrose [D-glucopyranosyl-alpha(1----5)-D-fructopyranose] as a sugar substitute.

Leucrose [D-glucosyl-alpha(1----5)-D-fructopyranose], prepared by an enzyme-catalyzed transglycosidation from sucrose with greater than 99% purity, has previously been shown to be noncariogenic and was found in the present study to be apparently easily digestible when given to humans as a single oral dose of 100 g, or when fed to rats at a level of 35 g/kg body wt daily. Weanling rats fed a 25% leucrose diet grew as well as rats fed a diet containing 25% sucrose or corn starch. When 1 g of leucrose was given intravenously to adult rats, 70% of this disaccharide was excreted in the urine within 24 h, and feeding and drinking behavior of the rats was not altered. No adverse effects on their general health were observed. The substrate properties of leucrose for alpha-glucosidase from yeast and for carbohydrases from human jejunal mucosa were determined, and these data were then compared with those of maltose and sucrose. The cleavage rate of leucrose in vitro by human digestive carbohydrases was 31% that of maltose and 63% that of sucrose. Hydrogenated leucrose (leucritol) was cleaved 10 times slower, with a Michaelis constant close to that of leucrose. Blood glucose and fructose profiles in humans given leucrose per os tended to be lower than those in humans given sucrose, while insulin and C-peptide profiles were unaltered.

Cariological assessment of leucrose [D-glucopyranosyl-alpha(1----5)-D-fructopyranose] as a sugar substitute.

Leucrose [D-glucosyl-alpha(1----5)-D-fructopyranose] prepared by microbial-enzymatic transglycosidation from sucrose, is the first alpha(1----5)-linked disaccharide which possesses excellent nutritional properties with regard to metabolic utilization and is well tolerated. The aim of the present work was to assess its cariogenic potential. Yeast invertase was shown to be inhibited by leucrose in a noncompetitive way, while hydrogenated leucrose (leucritol) acted as an activator. Plaque polysaccharide forming glucosyltransferases from Streptococcus cricetus AHT were not influenced by leucrose, but by leucritol. Essentially no acid formation was observed after incubation of leucrose with suspensions of human dental plaque, S. mutans NCTC 10449, Lactobacillus casei LSB 132 and Actinomyces viscosus Ny 1 No. 30. Leucrose was a competitive inhibitor of the acid formation from sucrose by S. mutans NCTC 10449 at neutral pH. Furthermore, leucrose inhibited at neutral pH considerably the uptake of sucrose by S. mutans NCTC 10449. The uptake of fructose and maltose was also inhibited but that of glucose not at all. In Cara rats as the animal model, leucrose was compared to sucrose and to corn starch for its cariogenic potential. In sharp contrast to the group fed with 30% sucrose, caries scores of the 30% leucrose group were not significantly different from the starch group. pH telemetry with an indwelling electrode in man proved lueucrose to be 'safe for teeth' since plaque pH did not drop below pH 5.7. Leucrose is a novel noncariogenic disaccharide and thus represents a highly promising sugar substitute for caries prevention.

Lactulose, 51Cr-labelled ethylenediaminetetra-acetate, L-rhamnose and polyethyleneglycol 400 [corrected] as probe markers for assessment in vivo of human intestinal permeability.

The urinary excretion of lactulose, 51Cr-labelled ethylenediaminetetra-acetate (51Cr-EDTA), L-rhamnose and polyethyleneglycol 400 (PEG-400) has been measured after intravenous and oral administration in healthy volunteers. Intestinal permeation of the probes was compared after their ingestion in iso-osmolar, hyperosmolar and cetrimide-containing test solutions. Urinary recovery of lactulose and 51Cr-EDTA after intravenous administration reached 75% by 5 h, and exceeded 90% at 24 h, and these values were 62 and 72%, respectively, for L-rhamnose. Recovery of PEG-400, however, varied with the relative molecular mass (Mr) of each polymer from 25.9 to 68.5% in 24 h. Intestinal permeation of ingested lactulose and 51Cr-EDTA was low, but that of L-rhamnose was 45-fold, and that of PEG-400 100-fold, greater. Permeation of lactulose and 51Cr-EDTA was markedly increased by cetrimide and hyperosmolar stress, whereas that of L-rhamnose showed little change. PEG-400 permeation was not affected by cetrimide, but was slightly increased by hyperosmolar stress. The 5 h permeation of lactulose, but not of L-rhamnose or PEG-400, correlated with that of 51Cr-EDTA (r = 0.98, P less than 0.001). These findings are compatible with three distinct pathways of unmediated mucosal permeation, L-rhamnose (radius less than 0.4 nm) passing mainly through small aqueous 'pores' of high incidence, lactulose and 51Cr-EDTA (radius greater than 0.5 nm) through larger aqueous 'channels' of low incidence susceptible to cetrimide and hyperosmolar stress, and PEG-400, which has appreciable lipid solubility, by partition through cell membrane lipid as well as the aqueous 'pores'.