The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes.

α-Synuclein is an intrinsically disordered presynaptic protein associated with Parkinson's disease. The physiological role of α-Synuclein is not fully understood, but the protein is known to interact with lipid membranes. We here study how membrane charge affects the adsorption of α-Synuclein to (i) supported lipid bilayers and (ii) small unilamellar vesicles with varying amounts of anionic lipids. The results showed that α-Synuclein adsorbs onto membranes containing ≥5% anionic phosphatidylserine (DOPS) lipids, but not to membranes containing ≤1% DOPS. The density of adsorbed α-Synuclein increased steadily with the DOPS content up to 20% DOPS, after which it leveled off. The vesicles were saturated with α-Synuclein at a 3-5 times higher protein density compared to the supported bilayers, which suggests that a more deformable membrane binds more α-Synuclein. Altogether, the results show that both membrane charge density and flexibility influence the association of α-Synuclein to lipid membranes.

Single-vesicle intensity and colocalization fluorescence microscopy to study lipid vesicle fusion, fission, and lipid exchange.

Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties.

Snapshot multicolor fluorescence imaging using double multiplexing of excitation and emission on a single detector.

Fluorescence-based multispectral imaging of rapidly moving or dynamic samples requires both fast two-dimensional data acquisition as well as sufficient spectral sensitivity for species separation. As the number of fluorophores in the experiment increases, meeting both these requirements becomes technically challenging. Although several solutions for fast imaging of multiple fluorophores exist, they all have one main restriction; they rely solely on spectrally resolving either the excitation- or the emission characteristics of the fluorophores. This inability directly limits how many fluorophores existing methods can simultaneously distinguish. Here we present a snapshot multispectral imaging approach that not only senses the excitation and emission characteristics of the probed fluorophores but also all cross term combinations of excitation and emission. To the best of the authors' knowledge, this is the only snapshot multispectral imaging method that has this ability, allowing us to even sense and differentiate between light of equal wavelengths emitted from the same fluorescing species but where the signal components stem from different excitation sources. The current implementation of the technique allows us to simultaneously gather 24 different spectral images on a single detector, from which we demonstrate the ability to visualize and distinguish up to nine fluorophores within the visible wavelength range.

Cooperativity of α-Synuclein Binding to Lipid Membranes.

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and µm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.

Elevated levels of kynurenic acid change the dopaminergic response to amphetamine: implications for schizophrenia.

Kynurenic acid (KYNA) is an endogenous compound implicated in the pathophysiology of schizophrenia. This tryptophan metabolite antagonizes both the N-methyl-D-aspartate (NMDA) receptors and the nicotinic alpha7* receptors at micromolar concentrations. In the present study the effects of amphetamine on dopamine (DA) release in the nucleus accumbens and on firing of DA neurons in the ventral tegmental area (VTA) were investigated in rats treated with kynurenine, the precursor of KYNA, in order to elevate brain KYNA levels. In rats subchronically treated with kynurenine (90 mg/kg x d for 6 d via osmotic minipumps, resulting in a 2-fold increase in whole-brain KYNA), the amphetamine-induced (2 mg/kg i.p.) increase in accumbal DA release was clearly enhanced compared to controls. Furthermore, subchronic treatment with kynurenine reduced the inhibitory action of amphetamine (0.2-25.6 mg/kg i.v.) on firing rate and burst firing activity of VTA DA neurons. A single dose of kynurenine (5 mg/kg s.c., 60 min, resulting in a 3-fold increase in whole-brain KYNA) did not alter the amphetamine-induced effects on DA neurotransmission compared to control rats. Present data are in agreement with the increased striatal DA release by amphetamine as observed by brain-imaging studies in patients with schizophrenia. Thus, subchronic elevation of rat brain KYNA, may rationally serve as an animal model similar to a pathophysiological condition of schizophrenia. It is proposed that the reduced responsivity of VTA DA neurons to the inhibitory action of amphetamine observed in rats with subchronically elevated KYNA levels may partly account for the increase in terminal DA release.

Activation of rat ventral tegmental area dopamine neurons by endogenous kynurenic acid: a pharmacological analysis.

Kynurenic acid (KYNA) is an endogenous NMDA receptor antagonist as well as a blocker of the alpha7* nicotinic receptor and mounting evidence suggests that the compound participates in the pathophysiology of schizophrenia. Previous studies have shown that elevated levels of endogenous KYNA are associated with an increased firing of midbrain dopamine (DA) neurons. In the present study, utilizing extracellular single unit cell recording techniques, the mechanism involved in this excitatory action of the compound was analyzed in male Sprague-Dawley rats. Administration of 4-chlorokynurenine (4-Cl-KYN; 25mg/kg, i.p.), which is converted to the selective NMDA glycine-site antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), was found to increase firing rate and per cent burst firing activity of ventral tegmental area (VTA) DA neurons to the same magnitude as pretreatment of kynurenine (causing a 25-fold elevation in extracellular brain KYNA). Intravenous administration of the selective antagonist at the alpha7* nicotinic receptor methyllycaconitine (MLA; 1-4mg/kg) did not affect firing of VTA DA neurons, whereas intraperitoneal administration of this drug in a high dose (6mg/kg) was associated with a decreased firing rate and per cent burst firing activity. Administration of SDZ 220-581 (10mg/kg, i.v.), a competitive antagonist at the glutamate recognition-site of the NMDA receptor, was found to increase firing rate and per cent burst firing. Present results have potential implications for the treatment of schizophrenia, and indicate that the increased activity of VTA DA neurons following elevation of brain KYNA is mediated through glutamatergic rather than by nicotinergic mechanisms.

Heat extraction of corn fiber hemicellulose.

Water-soluble hemicellulose was extracted from corn fiber with microwave-assisted heat treatment. The effects of treatment temperature and initial pH of the aqueous extraction media were investigated regarding hemicellulose recovery and molecular mass of the isolated polysaccharides. In treatments carried out at neutral pH (simple water extraction), it has been demonstrated that hemicellulose recovery could be increased by applying higher treatment temperatures. However, the molecular weight of isolated hemicellulose gets significantly lower. For example, 10% of the raw materials' xylan was extracted at 160 degrees C and about 30% recovery was reached at 210 degrees C. However, the molecular mass of the isolated polysaccharide at 210 degrees C (5.82 x 10(4)) was about half of that measured at 160 degrees C (1.37 x 10(5)). Reducing the pH with sulfuric acid resulted in shorter polymer chains (1.7 x 10(4)) and lower hemicellulose yields (2.2%). Application of sodium hydroxide in the treatment showed that, compared with acid, considerably higher yields (11%) with longer polysaccharide chains (1.3 x 10(5)) could be obtained.

Comparison of diafiltration and size-exclusion chromatography to recover hemicelluloses from process water from thermomechanical pulping of spruce.

Hemicelluloses constitute one of the most abundant renewable resources on earth. To increase their utilization, the isolation of hemicelluloses from industrial biomass side-streams would be beneficial. A method was investigated to isolate hemicelluloses from process water from a thermomechanical pulp mill. The method consists of three steps: removal of solids by microfiltration, preconcentration of the hemicelluloses by ultrafiltration, and purification by either size-exclusion chromatography (SEC) or diafiltration. The purpose of the final purification step is to separate hemicelluloses from small oligosaccharides, monosaccharides, and salts. The ratio between galactose, glucose, and mannose in oligo- and polysaccharides after preconcentration was 0.8:1:2.8, which is similar to that found in galactoglucomannan. Continuous diafiltration was performed using a composite fluoro polymer membrane with cutoff of 1000 Da. After diafiltration with four diavolumes the purity of the hemicelluloses was 77% (gram oligo- and polysaccharides/gram total dissolved solids) and the recovery was 87%. Purification by SEC was performed with 5, 20, and 40% sample loadings, respectively and a flow rate of 12 or 25 mL/min (9 or 19 cm/h). The purity of hemicelluloses after SEC was approx 82%, and the recovery was above 99%. The optimal sample load and flow rate were 20% and 25 mL/min, respectively. The process water from thermomechanical pulping of spruce is inexpensive. Thus, the recovery of hemicelluloses is not of main importance. If the purity of 77%, obtained with diafiltration, is sufficient for the utilization of the hemicelluloses, diafiltration probably offers a less expensive alternative in this application.

Affinity partitioning of a Cellulomonas fimi beta-mannanase with a mannan-binding module in galactomannan/starch aqueous two-phase system.

A new approach in affinity separations was studied by partitioning of Cellulomonas fimi beta-mannanase (EC 3.2.1.78) containing a mannan-binding module in galactomannan/hydroxypropyl starch aqueous two-phase system. Comparison was made with a truncated version of C. fimi beta-mannanase which lacked the mannan-binding module. Results showed that affinity partitioning of the beta-mannanase was achieved due to biospecificity of the mannan-binding module towards the top phase containing galactomannan. Experiments were conducted at pH 8 to prevent enzyme degradation of the phase containing galactomannan. Removal of the top phase polymer was accomplished by beta-mannanase degradation allowed by shifting to the optimal pH 6. In the combination with the genetic fusion of any given protein to the mannan-binding module, the results envision a general procedure for primary affinity recovery of such fusion proteins.