Lipid Release Research Articles

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.

When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids play both structural and regulatory roles. Here, we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting release of bioactive lipids from a critical allosteric regulatory site.

Lipid profiling of lipoprotein X: Implications for dyslipidemia in cholestasis

Lipoprotein X (Lp-X) is an abnormal lipoprotein that may typically be formed in intra- and extrahepatic cholestasis and potentially interfere with lipid analysis in the routine lab.To gain insight into lipid class and species composition, Lp-X, LDL and HDL from cholestatic and control serum samples were subjected to mass spectrometric analysis including phospholipids (PL), sphingolipids, free cholesterol (FC), cholesteryl esters (CE) and bile acids.Our analysis of Lp-X revealed a content of 46% FC, 49% PL with 34% phosphatidylcholine (PC) as main PL component. The lipid species pattern of Lp-X showed remarkable high fractions of mono-unsaturated species including PC 32:1 and PC 34:1 and phosphatidylethanolamine (PE) 32:1 and 34:1. LDL and HDL lipid composition in the same specimens strongly reflected the lipid composition of Lp-X with increased PC 32:1, PC 34:1, PE 32:1, PE 34:1 and FC accompanied by decreased CE compared to controls.Comparison of Lp-X and biliary lipid composition clearly indicates that Lp-X does not originate from a sole release of bile lipids. Moreover, these data present evidence for increased hepatic fatty acid and PL synthesis which may represent a reaction to high hepatic FC level observed during cholestasis.

Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores

Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered. From the lipid release times we computed pore openness, the fraction of time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested, SNARE-mediated and nonspecifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels, SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t-SNAREs to the fusion site, consistent with t-SNAREs being preclustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion-pore bending energy, and indirectly by concentrating several SNAREs into individual fusion events.

Predicting Initial Lipid Release from Masticated Tree Nuts Using Mathematical Modelling

IntroductionThe proportion of lipid released during mastication of nuts is strongly influenced by particle size, due to natural encapsulation of the lipid by the walls of intact cells. The cell walls may act as barriers to digestion and may partially explain why nuts have reduced metabolizable energy versus the energy content predicted by Atwater factors. The lipid released from masticated nuts can be calculated using a mathematical model which has the cell diameter and the particle size distribution (PSD) of the bolus as variables. This study measured the cell size and PSD of four tree nuts (raw cashews, raw walnuts, roasted pistachios and raw Brazil nuts) to predict the lipid released due to their mastication.ObjectivesTo predict the proportion of lipid released from masticated tree nuts, using measurements of cell size and PSD of masticated tree nuts (including cashews, walnuts, pistachios and Brazil nuts). To compare the results for these nuts to those already published for almonds.Method/DesignTransverse and longitudinal sections were cut from each nut (including almonds) and micrographs processed using image analysis software to calculate the average cell diameter. Two randomized, un‐blinded, cross‐over trials were conducted. In each trial, 10 healthy volunteers attended two sessions, at which they chewed eight samples of a randomly allocated nut. For determination of PSDs, the expectorated boluses were sieved (2 boluses) or analyzed by laser diffraction (2 boluses). Initial lipid release was then predicted using the mathematical model.ResultsThe cashew cells (34.3 μm) were smaller than the almond (45.1 μm), walnut (49.4 μm), pistachio (53.1 μm), and Brazil nut cells (60.8 μm). Laser diffraction showed that masticated nut boluses had median particle sizes (± SEM) which were smaller (cashews, 178 ± 12 μm; walnuts, 179 ± 8 μm; pistachios, 123 ± 10 μm; Brazil nuts, 145 ± 8 μm) than that for almonds measured previously (550 ± 18 μm). This results in higher predicted lipid release, calculated from the mathematical model, (mean, range) for cashews (12.3%, 8.7–16.3%), walnuts (14.5%, 12.0–18.0%), pistachios (11.0%, 8.7–13.0%) and Brazil nuts (14.4%, 11.9–17.3%) than for almonds (9.5%, 7.4–11.1%).ConclusionsAll of the five nuts had predicted lipid releases of less than 18%, which would be expected to attenuate postprandial lipemia and total nutrient availability relative to that expected due to their total lipid content. Due to their higher lipid content and predicted lipid release, walnuts and Brazil nuts (after mastication) are likely to release more fat on mastication than the other nuts tested.This research was approved by the relevant NHS National Research Ethics Committee (14/LO/2286 and 15/WS/0104) and registered at clinicaltrials.gov (NCT02398084 and NCT02497768).Support or Funding InformationThis research was funded by King's College London.

Overexpression of G0/G1 Switch Gene 2 in Adipose Tissue of Transgenic Quail Inhibits Lipolysis Associated with Egg Laying.

In avians, yolk synthesis is regulated by incorporation of portomicrons from the diet, transport of lipoproteins from the liver, and release of lipids from adipose tissue; however, the extent to which lipolysis in adipose tissue contributes to yolk synthesis and egg production has yet to be elucidated. G0/G1 switch gene 2 (G0S2) is known to bind and inhibit adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis. The objective of this study was to determine whether overexpression of the G0S2 gene in adipose tissue could successfully inhibit endogenous ATGL activity associated with egg laying. Two independent lines of transgenic quail overexpressing G0S2 had delayed onset of egg production and reduced number of eggs over a six-week period compared to non-transgenic quail. Although no differences in measured parameters were observed at the pre-laying stage (5 weeks of age), G0S2 transgenic quail had significantly larger interclavicular fat pad weights and adipocyte sizes and lower NEFA concentrations in the serum at early (1 week after laying first egg) and active laying (5 weeks after laying first egg) stages. Overexpression of G0S2 inhibited lipolysis during early and active laying, which drastically shifted the balance towards a net accumulation of triacylglycerols and increased adipose tissue mass. Thereby, egg production was negatively affected as less triacylglycerols were catabolized to produce lipids for the yolk.

Encapsulation of curcumin in polysaccharide-based hydrogel beads: Impact of bead type on lipid digestion and curcumin bioaccessibility

Curcumin was incorporated into three different delivery systems: free lipid droplets; lipid-loaded alginate beads; and, lipid-loaded carrageenan beads. Hydrogel beads were fabricated from polysaccharides (alginate or carrageenan) using an injection method combined with ion gelation (calcium or potassium, respectively). The delivery systems were passed through a simulated gastrointestinal tract (GIT) that included mouth, stomach, and small intestine phases. Light scattering and microscopy indicated that carrageenan beads had a relatively fragile structure that was easily disrupted in the GIT and released the encapsulated lipid droplets and curcumin. Conversely, alginate beads had a robust structure that remained relatively intact throughout the GIT and retained the lipid droplets and curcumin. The rate and extent of lipid digestion decreased in the following order: free lipid droplets > carrageenan beads > alginate beads. Curcumin bioaccessibility followed a similar order: free lipid droplets (73%) > carrageenan beads (33%) > alginate beads (16%). These results suggest that lipid droplets must be digested by lipase in order to release the encapsulated curcumin and to form mixed micelles capable of solubilizing the released curcumin. Overall, our results show that delivery systems with different structures and compositions can be designed to control the release of lipids and hydrophobic nutraceuticals in the GIT, which may be advantageous for the development of certain functional food products.

How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast.

In plants, the products of secretory activity leave the protoplast and cross the plasma membrane by means of transporters, fusion with membranous vesicles or, less commonly, as result of disintegration of the cell. These mechanisms do not address an intriguing question: How do secretory products cross the cell wall? Furthermore, how do these substances reach the external surface of the plant body? Such diverse substances as oils, polysaccharides or nectar are forced to cross the cell wall and, in fact, do so. How are chemical materials that are repelled by the cell wall or that are sufficiently viscous to not cross passively released from plant cells? I propose a cell-cycle model developed based on observations of different secreting systems, some unpublished results and an extensive literature review, aiming to understand the processes involved in both the secretory process and the release of secretion products. In the absence of facilitated diffusion, a mechanical action of the protoplast is necessary to ensure that some substances can cross the cell wall. The mechanical action of the protoplast, in the form of successive cycles of contraction and expansion, causes the material accumulated in the periplasmic space to cross the cell wall and the cuticle. This action is particularly relevant for the release of lipids, resins and highly viscous hydrophilic secretions. The proposed cell-cycle model and the statements regarding exudate release will also apply to secretory glands not elaborated upon here. Continuous secretion of several days, as observed in extrafloral nectaries, salt glands and some mucilage-producing glands, is only possible because the process is cyclical.

Effect of spice-incorporated starch edible film wrapping on shelf life of white shrimps stored at different temperatures.

White shrimps (Litopenaeus vannamei) are a major aquaculture product in the world fishery market. The main aim of this study was to investigate the effect of clove- and cinnamon-assimilated starch edible films on the shelf life of white shrimps in terms of maintaining their freshness and other organoleptic properties. Physical, chemical, microbial and sensory qualities of edible film-wrapped white shrimps were studied until they reached their limit of acceptability during storage at different temperatures (10 and 4 °C). Shrimp samples wrapped with spice-assimilated edible films showed lower bacterial counts. Shelf life extension of edible film-wrapped white shrimps was estimated to be 14 and 12 days for storage at 10 and 4 °C respectively. Reduced lipid oxidation and release of nitrogen base compounds were noted for edible film-wrapped shrimp samples. Good consumer acceptance was noted for edible film-wrapped shrimp samples through sensory evaluation. The results of this study show that spice-fused edible films were effective in inhibiting the growth of microbial populations. Reductions in lipid oxidation and total volatile base nitrogen were also achieved through edible film wrapping of shrimps, which increased their consumer acceptance during sensory evaluation. © 2016 Society of Chemical Industry.

SNARE-Mediated Transient Fusion of Liposomes to Supported Bilayers Probed by Two-Color pTIRFM

In membrane fusion opening of a fusion pore establishes the first connection between two compartments. During neurotransmitter or hormone release via exocytosis, fusion pores can transiently open and close repeatedly (flicker) before resealing (‘kiss-and-run’) or dilating irreversibly (full fusion). Pore dynamics regulate the amount and size of released cargo and determine the mode of vesicle recycling, yet what governs pore dynamics is poorly understood. We established an assay to monitor membrane fusion by polarized total internal reflection (pTIRF) microscopy with single molecule sensitivity and ∼15 ms time resolution in a biochemically well-defined system. In our assay, fusion of small unilamellar vesicles containing v-SNARE proteins (v-SUVs) fuse to a flat bilayer reconstituted with cognate t-SNAREs, supported on a soft polymer cushion (t-SBL). Exploiting the signal enhancement when a label transfers from the SUV to the SBL membrane in pTIRF, we could monitor the release kinetics of vesicular lipid labels into the SBL upon fusion. We found release times were much longer than would be expected for permanently open pores. Our sensitivity to single fluorescent lipids allowed us to calculate the lipid diffusivity and SUV size for each fusion event. This was used in a model we developed assuming the retardation was due to flickering pores, to estimate pore openness P0, the fraction of time a pore remains open during fusion. Increasing cholesterol levels increased P0. However, retardation of lipid release could also be due to steric hindrance of lipid diffusion through the walls of the fusion pore by SNARE proteins. To exclude this, we encapsulated soluble markers into the SUVs and simultaneously monitored lipid and contents release. Release of both lipid and soluble cargo started simultaneously. Interestingly, many pores resealed after partially releasing their contents.

Method to disperse lipids as aggregates in oil for bilayers production.

Several techniques to assemble artificial lipid bilayers involve the zipping of monolayers. Their efficiency is determined by the renewal of the saturated monolayers to be zipped and this proceeds by adsorption of lipids dispersed in oil as aggregates. The size of these lipids aggregates is a key parameter to ensure both the stability of the suspension and a fast release of lipids at the interface. We propose a new method inspired from the solvent-shifting nucleation process allowing to control and tune the lipid aggregates size and that improves the production of artificial membranes. It is simpler and faster than current methods starting from a dry lipid film, which are highly sensitive to environmental conditions. This method opens the route to bilayer production processes with new potentialities in membrane composition.

Effect of the N-Terminal Helix and Nucleotide Loading on the Membrane and Effector Binding of Arl2/3

The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.

Determination of Vitamin K1 in Infant, Pediatric, and Adult Nutritionals by HPLC with Fluorescence Detection: Single-Laboratory Validation, First Action 2015.09.

This normal-phase HPLC method with postcolumn reduction and fluorescence detection allows for the quantitative determination of trans vitamin K1 in infant, pediatric, and adult nutritionals. Vitamin K1 is extracted from products with iso-octane after precipitation of proteins and release of lipids with methanol. Prepared samples are injected onto a silica HPLC column where cis and trans vitamin K1 are separated with an iso-octane-isopropanol mobile phase. The column eluent is mixed with a dilute ethanolic solution of zinc chloride, sodium acetate, and acetic acid, and vitamin K1 is reduced to a fluorescent derivative in a zinc reactor column. The resulting hydroquinone is then detected by fluorescence at an excitation wavelength of 245 nm and an emission wavelength of 440 nm. During a single-laboratory validation of this method, repeatability and intermediate precision ranged from 0.6 to 3.5% RSD and 1.1 to 6.0% RSD, respectively. Mean overspike recoveries ranged from 91.9 to 106%. The method demonstrated good linearity over a standard range of approximately 2-90 μg/L trans vitamin K1 with r2 averaging 0.99995 and average calibration errors of <1%. LOQ and LOD in ready-to-feed nutritionals were estimated to be 0.03 and 0.09 μg/100 g, respectively. The method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements® and was approved as a first action method at the 2015 AOAC Mid-Year Meeting.

ADVANCES IN PHARMACOLOGICAL AND PHYTOCHEMICAL ADIPOGENESIS THERAPY

The main function of the adipocyte is lipid storage when there is a positive energy balance and lipid release when there is and energy deficiency. One characteristic of obesity is an increase in the number and size of adipocytes, which implies pre adipocyte (PAD) differentiation. The adipose tissue (AT) has its origins in the prenatal stage and may continue to expand during adulthood from precursor cells since mature adipocytes cannot multiply by cell division. This study provide updates on the events that occur during the origin and differentiation of PAD, the factors involved in the regulation of adipogenesis and mechanisms that regulate physiological functions of AT.

Thermal transitions in serum amyloid A in solution and on the lipid: implications for structure and stability of acute-phase HDL

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.

Roles of Chemokines and Chemokine Receptors in Obesity-Associated Insulin Resistance and Nonalcoholic Fatty Liver Disease.

Abundant evidence has demonstrated that obesity is a state of low-grade chronic inflammation that triggers the release of lipids, aberrant adipokines, pro-inflammatory cytokines, and several chemokines from adipose tissue. This low-grade inflammation underlies the development of insulin resistance and associated metabolic comorbidities such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). During this development, adipose tissue macrophages accumulate through chemokine (C-C motif) receptor 2 and the ligand for this receptor, monocyte chemoattractant protein-1 (MCP-1), is considered to be pivotal for the development of insulin resistance. To date, the chemokine system is known to be comprised of approximately 40 chemokines and 20 chemokine receptors that belong to the seven-transmembrane G protein-coupled receptor family and, as a result, chemokines appear to exhibit a high degree of functional redundancy. Over the past two decades, the physiological and pathological properties of many of these chemokines and their receptors have been elucidated. The present review highlights chemokines and chemokine receptors as key contributing factors that link obesity to insulin resistance, T2DM, and NAFLD.

When fat becomes an ally of the enemy: adipose tissue as collaborator in human breast cancer.

Since the discovery of leptin in 1994, our vision of adipose tissue as a static organ regulating mainly lipid storage and release has been completely overthrown, and adipose tissue is now seen as an active and integral organ in human physiology. In the past years, extensive research has tremendously given us more insights in the mechanisms and pathways involved not only in normal but also in 'sick' adipose tissue, for example, in obesity and lipodystrophy. With growing evidence of a link between obesity and several types of cancer, research focusing on the interaction between adipose tissue and cancer has begun to unravel the interesting but complex multi-lateral communication between the different players. With breast cancer as one of the first cancer types where a positive correlation between obesity and breast cancer incidence and prognosis in post-menopausal women was found, we have focused this review on the paracrine and endocrine role of adipose tissue in breast cancer initiation and progression. As important inter-species differences in adipose tissue occur, we mainly selected human adipose tissue- and breast cancer-based studies with a short reflection on therapeutic possibilities. This review is part of the special issue on "Adiposopathy in Cancer and (Cardio)Metabolic Diseases".

Time Scales of Conformational Gating in a Lipid-Binding Protein.

Lipid-binding proteins sequester amphiphilic molecules in a large internal cavity occupied by ∼30 water molecules, some of which are displaced by the ligand. The role of these internal water molecules in lipid binding and release is not understood. We use magnetic relaxation dispersion (MRD) to directly monitor internal-water dynamics in apo and palmitate-bound rat intestinal fatty acid-binding protein (rIFABP). Specifically, we record the water (2)H and (17)O MRD profiles of the apo and holo forms of rIFABP in solution or immobilized by covalent cross-links. A global analysis of this extensive data set identifies three internal-water classes with mean survival times of ∼1 ns, ∼100 ns, and ∼6 μs. We associate the two longer time scales with conformational fluctuations of the gap between β-strands D and E (∼6 μs) and of the portal at the helix-capped end of the β-barrel (∼100 ns). These fluctuations limit the exchange rates of a few highly ordered structural water molecules but not the dissociation rate of the fatty acid. The remaining 90% (apo) or 70% (holo) of cavity waters exchange among internal hydration sites on a time scale of ∼1 ns but exhibit substantial orientational order, particularly in the holo form.

Effects of gallium, indium, and arsenic dose biomarkers and malondialdehyde on zinc protoporphyrin and DNA fragments in optoelectronic workers

Purpose: To investigate whether occupational exposure to gallium, indium, and arsenic may disrupt normal heme production and damage DNA by initiating the release of lipid peroxidation.Methods: 101exposed optoelectronic workers and 65 referents were tested for their whole blood and urine levels of gallium, indium, and arsenic. Plasma malondialdehyde (MDA), blood zinc protophorphyrin (ZPP), and serum DNA fragments were used as indicators of toxic hazards.Results: The MDA, ZPP, and DNA fragment mean levels in the exposed workers were significantly higher than those in the referents. The levels of urine gallium in the exposed workers were significantly correlated with the levels of whole blood ZPP and serum DNA fragments respectively. MDA (the production of lipid peroxidation) may be one of the mechanisms for gallium-related DNA damage in multiple regression models. In addition, gallium was found to have a synergistic effect with arsenic on MDA level that may induce indium to damage DNA in blood cells.Conclusion: ZPP and DNA fragments can be elevated by gallium exposure, and the MDA increase due to combined exposure to gallium and arsenic may play a role in indium-induced DNA damage.