Behavioral discrimination of binary mixtures and their components: effects of mixture interactions on coding of stimulus intensity and quality

There is mounting evidence that mixture interactions resultin a physiological response that is different from that predictedfrom observed responses to individual mixture components. Mixtureinteractions that act to alter the neural coding of mixtureintensity (intensity mixture interactions) or quality (patternmixture interactions) may ultimately lead to dramatic differencesbetween the perceived intensities and qualities of a mixtureand its components. These perceptions could be expressed andobserved at the behavioral level. Toward examining this question,we have tested the ability of the Florida spiny lobster (Panulirusargus) to behaviorally discriminate between three odorant compounds[adenosine 5'-monophosphate (AMP), L-glutamate (Glu), and taurine(Tau)] and their binary mixtures through the use of a differentialaversive associative conditioning paradigm. Six groups of lobsterswere used, each being conditioned to avoid one of the singlecompounds or binary mixtures. Behavioral expression of intensitymixture interactions was evident. Preconditioning response magnitudesto binary mixtures were either less than those to their components(e.g. AMP + Glu) or less than predicted from responses to theircomponents (e.g. AMP + Tau). Behavioral expression of patternmixture interactions was also observed. Relationships betweenthe quality of each binary mixture and the qualities of themixture's components were determined from the results of analysisof variance and multidimensional scaling analysis. Analysesincorporated observed responses to all stimuli and ‘predicted’responses to the binary mixtures. Lobsters easily discriminatedbetween the qualities of AMP, Glu and Tau. The quality of themixture of AMP + Glu was different from either component aswell as from the predicted value for this mixture. The mixtureof AMP + Tau was intermediate between both components and wassimilar to the predicted value. The mixture of Glu + Tau, whilemore similar to Glu than to Tau, was different from the predictedvalue, and there was some indication that the Glu was actingto suppress the response to Tau. Behavioral results for AMP+ Tau, which suggest no pattern mixture interactions betweenthese compounds, are in accordance with results of recentlyconducted binding assays which indicate independent receptorsfor these compounds (Olson et al., 1992). Results, especiallyfor AMP + Glu and Glu + Tau, are consistent with results ofour electrophysiological analysis of the effects of patternmixture interactions on coding of stimulus quality and intensityby olfactory receptor cells (Derby et al., 1991a,b). This providesfurther evidence for the effects of peripherally initiated mixtureinteractions on the coding and perception of the quality ofodorant mixtures. ¹Present address: Departments of Psychology and Biology, GeorgiaState University, University Plaza, Atlanta, Georgia 30303,USA     

Complex binding interactions between multicomponent mixtures and odorant receptors in the olfactory organ of the Caribbean spiny lobster Panulirus argus

Our study was designed to examine how components of complex mixtures can inhibit the binding of other components to receptor sites in the olfactory system of the spiny lobster Panulirus argus. Biochemical binding assays were used to study how two- to six-component mixtures inhibit binding of the radiolabeled odorants taurine, L-glutamate and adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane of olfactory receptor neurons. Our results indicate that binding inhibition by mixtures can be large and is dependent on the nature of the odorant ligand and on the concentration and composition of the mixture. The binding inhibition by mixtures of structurally related components was generally predicted using a competitive binding model and binding inhibition data for the individual components. This was not the case for binding inhibition by most mixtures of structurally unrelated odorants. The binding inhibition for these mixtures was generally smaller than that for one or more of their components, indicating that complex binding interactions between components can reduce their ability to inhibit binding. The magnitude of binding inhibition was influenced more by the mixture's precise composition than by the number of components in it, since mixtures with few components were sometimes more inhibitory than mixtures with more components. These findings raise the possibility that complex binding interactions between components of a mixture and their receptors may shape the output of olfactory receptor neurons to complex mixtures.      

Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.     

Some implications of a first-order model of inter-plant competition for the means and variances of complex mixtures

Summary Factorial models commonly used in the analysis of overall and component yields of binary mixtures of genotypes are generalised to include mixtures of any number of components (size, m) and the form of an analysis of variance for fitting such a model to tertiary mixtures is outlined. Such a model contains main effects and interactions up to the mth order, and is specific to the size of mixture so that no equivalence necessarily exists between similar parameter sets for different sized mixtures. Monocultures can be regarded as a special case of the general model.A simple model of intra-and inter-component competition is defined which assumes that plants do not interact in their competitive effects on others, a condition which is equivalent to an absence of second and higher order interactions in statistical analyses of mixtures of any size. Simple scaling tests involving the yields of components or whole mixtures of different sizes can also be used to test the adequacy of the model. This competition model least to a linear relationship between the mean yield of a mixture and the reciprocal of the number of components it contains, and thus allows the prediction of means and other statistical parameters for mixtures of one size from those of others.     

Experimental and computational studies of enantioseparation of structurally similar chiral compounds on amylose tris(3,5‐dimethylphenylcarbamate)

The enantioseparation of 14 structurally similar chiral solutes, with one or two chiral centers, are studied for a commercially important polysaccharide‐based chiral stationary phase, amylose tris(3,5‐dimethylphenylcarbamate) (ADMPC). Among these solutes, only two solutes show significant enantioresolutions of 2 to 2.5 in n‐hexane/2‐propanol (90/10, v/v) at 298 K. The retention factors of the chiral solutes vary significantly from 0.7 to 7.0, and they are compared with those of simpler nonchiral solutes having similar but fewer functional groups. The sorbent–solute H‐bonding interactions between the solute functional groups and the polymer C?O and NH functional groups are probed with attenuated total reflection infrared spectroscopy (ATR‐IR). The H‐bonding interactions of the polymer C?O and NH groups with the solutes result in changes in the IR amide band wavenumbers of ADMPC upon solute adsorption. The nanostructure of an ADMPC cavity and the potential interactions with the chiral solutes are proposed based on the sorbent–solute–solvent HPLC data, the sorbent–solute IR data, and the sorbent–solute molecular dynamics (MD) simulations. The results are consistent with the three point attachment hypothesis and indicate that a significant enantioresolution in ADMPC requires at least three different interaction sites for simultaneous H‐bonds and phenyl–phenyl interactions for phenylpropylamine (PPA) and various structurally similar chiral solutes. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Two Distinct Mechanisms of Transport Through the Plasmodial Surface Anion Channel

The plasmodial surface anion channel (PSAC) is a voltage-dependent ion channel on erythrocytes infected with malaria parasites. To fulfill its presumed function in parasite nutrient acquisition, PSAC is permeant to a broad range of charged and uncharged solutes; it nevertheless excludes Na⁺ as required to maintain erythrocyte osmotic stability in plasma. Another surprising property of PSAC is its small single-channel conductance (<3 pS in isotonic Cl^?) in spite of broad permeability to bulky solutes. While exploring the mechanisms underlying these properties, we recently identified interactions between permeating solutes and PSAC inhibitors that suggest the channel has more than one route for passage of solutes. Here, we explored this possibility with 22 structurally diverse solutes and found that each could be classified into one of two categories based on effects on inhibitor affinity, the temperature dependence of these effects and a clear pattern of behavior in permeant solute mixtures. The clear separation of these solutes into two discrete categories suggests two distinct mechanisms of transport through this channel. In contrast to most other broad-permeability channels, selectivity in PSAC appears to be complex and cannot be adequately explained by simple models that invoke sieving through rigid, noninteracting pores.     

Activity of Azole Fungicides and ABC Transporter Modulators on Mycosphaerella graminicola

The antimicrobial activity of the azole fungicides cyproconazole and propiconazole as single active ingredients and in mixtures with the ATP-Binding Cassette (ABC) transporter modulators rhodamine 6G, quercetin, quinidine, and verapamil and the strobilurin kresoxim-methyl was assessed against the wheat pathogen Mycosphaerella graminicola . Interactions amongst these compounds were evaluated on germination and germ tube growth of pycnidiospores using the Colby and Wadley method. Water agar proved to be the best test medium since all pycnidiospores germinated within 24 h of incubation and apical germ tube growth dominated over bud formation by intermediate cells. Analysis with the Colby method revealed that interactions between the compounds in all mixtures tested on germination of pycnidiospores were additive. With regard to germ tube growth, mixtures of cyproconazole and verapamil or kresoxim-methyl displayed a synergistic interaction. Analysis of mixtures of cyproconazole and kresoxim-methyl with the Wadley method revealed that the interaction between the two compounds was purely additive. These results indicate that the Colby method overestimated the interaction between these two compounds in a mixture.     

Effect of the solute molecular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate)

The effects of the molecular structures for 13 structurally similar chiral solutes on their HPLC retention and enantioresolutions on a commercially important polysaccharide-based chiral stationary phase, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied. Among these 13 solutes, only methyl ephedrine (MEph) shows significant enantioresolution. The retention factors of these chiral solutes vary significantly from 0.7 to 3.2 in n-hexane/2-propanol (90/10, v/v) at 298 K. The retention factors of some simpler non-chiral solutes having similar but fewer functional groups than their chiral counterparts are also studied under the same conditions and are compared to those of the chiral solutes. The H-bonding interactions between the functional groups of the solute and the C=O and NH functional groups of the polymer are probed with attenuated total reflection-infrared spectroscopy (ATR-IR) for the polymer, for binary sorbent-solute systems. The CDMPC IR amide band wavenumbers change significantly, indicating H-bonding interactions of the polymer C=O and NH groups with the solutes. The elution orders predicted for the enantiomers of these chiral solutes using molecular dynamics (MD) simulations of the polymer-solute binary systems are consistent with the HPLC results. The CDMPC cavity nano-structure and the potential interactions with chiral solutes are proposed based on HPLC data, IR data, and the simulations. The results are consistent with the three-point attachment model and support the hypothesis that significant enantioresolution requires at least three different synergistic interactions which can be a combination of steric hindrance, H-bonding, or pi-pi interactions.     

Discovery of aminopiperidine-based Smac mimetics as IAP antagonists

A series of structurally unique Smac mimetics that act as antagonists of inhibitor of apoptosis proteins (IAPs) has been discovered. While most previously described Smac mimetics contain the proline ring (or a similar cyclic motif) found in Smac, a key feature of the compounds described herein is that this ring has been removed. Despite this, compounds in this series potently bind to cIAP1 and elicit the expected phenotype of cIAP1 inhibition in cancer cells. Marked selectivity for cIAP1 over XIAP is observed for these compounds, which is attributed to a slight difference in the binding groove between the two proteins and the resulting steric interactions with the inhibitors. XIAP binding can be improved by constraining the inhibitor so that these unfavorable steric interactions are minimized.     

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.     

Self-assembly processes in the prebiotic environment

An important question guiding research on the origin of life concerns the environmental conditions where molecular systems with the properties of life first appeared on the early Earth. An appropriate site would require liquid water, a source of organic compounds, a source of energy to drive polymerization reactions and a process by which the compounds were sufficiently concentrated to undergo physical and chemical interactions. One such site is a geothermal setting, in which organic compounds interact with mineral surfaces to promote self-assembly and polymerization reactions. Here, we report an initial study of two geothermal sites where mixtures of representative organic solutes (amino acids, nucleobases, a fatty acid and glycerol) and phosphate were mixed with high-temperature water in clay-lined pools. Most of the added organics and phosphate were removed from solution with half-times measured in minutes to a few hours. Analysis of the clay, primarily smectite and kaolin, showed that the organics were adsorbed to the mineral surfaces at the acidic pH of the pools, but could subsequently be released in basic solutions. These results help to constrain the range of possible environments for the origin of life. A site conducive to self-assembly of organic solutes would be an aqueous environment relatively low in ionic solutes, at an intermediate temperature range and neutral pH ranges, in which cyclic concentration of the solutes can occur by transient dry intervals.     

The influence of sodium salts on binary mixtures of bitter-tasting compounds

In order to study potential mixture interactions among bitter compounds, selected sodium salts were added to five compounds presented either alone or as binary bitter-compound mixtures. Each compound was tested at a concentration that elicited 'weak' perceived bitterness. The bitter compounds were mixed at these concentrations to form a subset of possible binary mixtures. For comparison, the concentration of each solitary compound was doubled to measure bitterness inhibition at the higher intensity level elicited by the mixtures. The following sodium salts were tested for bitterness inhibition: 100 mM sodium chloride (salty), 100 mM sodium gluconate (salty), 100 and 20 mM monosodium glutamate (umami), and 50 mM adenosine monophosphate disodium salt (umami). Sucrose (sweet) was also employed as a bitterness suppressor. The sodium salts differentially suppressed the bitterness of compounds and their binary combinations. Although most bitter compounds were suppressed, the bitterness of tetralone was not suppressed, nor was the bitterness of the binary mixtures that contained it. In general, the percent suppression of binary mixtures of compounds was predicted by the average percent suppression of its two components. Within the constraints of the present study, the bitterness of mixtures was suppressed by sodium salts and sucrose independently, with few bitter interactions. This is consistent with observations that the bitter taste system integrates the bitterness of multi-compound solutions linearly.     

Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract.

A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions.     

Differential dimethyl labeling of N-termini of peptides after guanidination for proteome analysis

We describe an enabling technique for proteome analysis based on isotope-differential dimethyl labeling of N-termini of tryptic peptides followed by microbore liquid chromatography (LC) matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS). In this method, lysine side chains are blocked by guanidination to prevent the incorporation of multiple labels, followed by N-terminal labeling via reductive amination using d(0),(12)C-formaldehyde or d(2),(13)C-formaldehyde. Relative quantification of peptide mixtures is achieved by examining the MALDI mass spectra of the peptide pairs labeled with different isotope tags. A nominal mass difference of 6 Da between the peptide pair allows negligible interference between the two isotopic clusters for quantification of peptides of up to 3000 Da. Since only the N-termini of tryptic peptides are differentially labeled and the a(1) ions are also enhanced in the MALDI MS/MS spectra, interpretation of the fragment ion spectra to obtain sequence information is greatly simplified. It is demonstrated that this technique of N-terminal dimethylation (2ME) after lysine guanidination (GA) or 2MEGA offers several desirable features, including simple experimental procedure, stable products, using inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separation. LC-MALDI MS combined with this 2MEGA labeling technique was successfully used to identify proteins that included polymorphic variants and low abundance proteins in bovine milk. In addition, by analyzing a mixture of two equal amounts of milk whey fraction as a control, it is shown that the measured average ratio for 56 peptide pairs from 14 different proteins is 1.02, which is very close to the theoretical ratio of 1.00. The calculated percentage error is 2.0% and relative standard deviation is 4.6%.     

The Application of Gaussian Mixture Models for Signal Quantification in MALDI-ToF Mass Spectrometry of Peptides

Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) coupled with stable isotope standards (SIS) has been used to quantify native peptides. This peptide quantification by MALDI-TOF approach has difficulties quantifying samples containing peptides with ion currents in overlapping spectra. In these overlapping spectra the currents sum together, which modify the peak heights and make normal SIS estimation problematic. An approach using Gaussian mixtures based on known physical constants to model the isotopic cluster of a known compound is proposed here. The characteristics of this approach are examined for single and overlapping compounds. The approach is compared to two commonly used SIS quantification methods for single compound, namely Peak Intensity method and Riemann sum area under the curve (AUC) method. For studying the characteristics of the Gaussian mixture method, Angiotensin II, Angiotensin-2-10, and Angiotenisn-1-9 and their associated SIS peptides were used. The findings suggest, Gaussian mixture method has similar characteristics as the two methods compared for estimating the quantity of isolated isotopic clusters for single compounds. All three methods were tested using MALDI-TOF mass spectra collected for peptides of the renin-angiotensin system. The Gaussian mixture method accurately estimated the native to labeled ratio of several isolated angiotensin peptides (5.2% error in ratio estimation) with similar estimation errors to those calculated using peak intensity and Riemann sum AUC methods (5.9% and 7.7%, respectively). For overlapping angiotensin peptides, (where the other two methods are not applicable) the estimation error of the Gaussian mixture was 6.8%, which is within the acceptable range. In summary, for single compounds the Gaussian mixture method is equivalent or marginally superior compared to the existing methods of peptide quantification and is capable of quantifying overlapping (convolved) peptides within the acceptable margin of error.     

Effects of protein perturbants on phospholipid bilayers

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.     

Detection of two-component mixtures of lognormal distributions in grouped, doubly truncated data: analysis of red blood cell volume distributions

We have examined the statistical requirements for the detection of mixtures of two lognormal distributions in doubly truncated data when the sample size is large. The expectation-maximization algorithm was used for parameter estimation. A bootstrap approach was used to test for a mixture of distributions using the likelihood ratio statistic. Analysis of computer simulated mixtures showed that as the ratio of the difference between the means to the minimum standard deviation increases, the power for detection also increases and the accuracy of parameter estimates improves. These procedures were used to examine the distribution of red blood cell volume in blood samples. Each distribution was doubly truncated to eliminate artifactual frequency counts and tested for best fit to a single lognormal distribution or a mixture of two lognormal distributions. A single population was found in samples obtained from 60 healthy individuals. Two subpopulations of cells were detected in 25 of 27 mixtures of blood prepared in vitro. Analyses of mixtures of blood from 40 patients treated for iron-deficiency anemia showed that subpopulations could be detected in all by 6 weeks after onset of treatment. To determine if two-component mixtures could be detected, distributions were examined from untransfused patients with refractory anemia. In two patients with inherited sideroblastic anemia a mixture of microcytic and normocytic cells was found, while in the third patient a single population of microcytic cells was identified. In two family members previously identified as carriers of inherited sideroblastic anemia, mixtures of microcytic and normocytic subpopulations were found. Twenty-five patients with acquired myelodysplastic anemia were examined. A good fit to a mixture of subpopulations containing abnormal microcytic or macrocytic cells was found in two. We have demonstrated that with large sample sizes, mixtures of distributions can be detected even when distributions appear to be unimodal. These statistical techniques provide a means to characterize and quantify alterations in erythrocyte subpopulations in anemia but could also be applied to any set of grouped, doubly truncated data to test for the presence of a mixture of two lognormal distributions.     

Compatible solute biosynthesis in cyanobacteria

Compatible solutes are a functional group of small, highly soluble organic molecules that demonstrate compatibility in high amounts with cellular metabolism. The accumulation of compatible solutes is often observed during the acclimation of organisms to adverse environmental conditions, particularly to salt and drought stress. Among cyanobacteria, sucrose, trehalose, glucosylglycerol and glycine betaine are used as major compatible solutes. Interestingly, a close correlation has been discovered between the final salt tolerance limit and the primary compatible solute in these organisms. In addition to the dominant compatible solutes, many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline as secondary or tertiary solutes. In particular, the accumulation of sucrose and trehalose results in an increase in tolerance to general stresses such as desiccation and high temperatures. During recent years, the biochemical and molecular basis of compatible solute accumulation has been characterized using cyanobacterial model strains that comprise different salt tolerance groups. Based on these data, the distribution of genes involved in compatible solute synthesis among sequenced cyanobacterial genomes is reviewed, and thereby, the major compatible solutes and potential salt tolerance of these strains can be predicted. Knowledge regarding cyanobacterial salt tolerance is not only useful to characterize strain-specific adaptations to ecological niches, but it can also be used to generate cells with increased tolerance to adverse environmental conditions for biotechnological purposes.     

Decomposition of spruce litter needles of different quality by Setulipes androsaceus and Thysanophora penicillioides

Different components of functional biodiversity, such as functional type richness and composition, have been reported to affect the decomposition of litter mixtures. In spite of the numerous reports of these effects, mechanisms underlying patterns of decomposition in litter mixtures are still unclear. We analyzed whether mixture decomposition was affected by: (a) the number of species in the mixture (mixture richness); and (b) the mixture’s functional composition (% of fast- vs. slow-decomposing species included in the mixture). We then tested if variation between observed and expected values of decomposition in mixtures was associated to: (c) the initial litter characteristics of the component species (initial nitrogen, lignin, cellulose and hemicellulose content of litters); and (d) the chemical heterogeneity of the mixtures (variation in the same chemical compounds between the components in each mixture). When up to 5 species representing different functional types were included, both species richness and functional composition showed statistically significant non-additive, and in general positive, effects on litter mixture decomposition. The positive effect of mixture richness on decomposition did not disappear, but was much less marked, when considering mixture with slow-decomposing species only. Although the main driver of decomposition in a mixture is still the average decomposability of the component species (itself largely determined by litter quality), the species interactions in a mixture add a consistent source of variability that is worth considering when predicting the decomposability of a given mixture. We showed that a greater positive difference between observed decomposition rates and that expected from its component species alone was found in mixtures with higher mean nitrogen content and a higher heterogeneity in non-labile compounds. Our results offer quantitative proof that litter chemical heterogeneity, as well as the mean quality of the mixture, can affect the decomposability in litter mixtures.