Ocular Amyloid, Condensates, and Aggregates - Higher-Order Protein Assemblies Participate in Both Retinal Degeneration and Function.

The formation of higher-order protein assemblies (commonly called protein aggregates) has long been associated with disease states, particularly in neurodegenerative disorders. Within the eye, protein aggregation has also been implicated in various retinal degenerative diseases ranging from retinitis pigmentosa (RP) to Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) to age-related macular degeneration (AMD). Yet, many essential cellular processes including transcription, translation, and the formation of non-membrane bound organelles require the formation of functional, non-pathologic protein aggregates to maintain cellular homeostasis. Thus, functional protein aggregates, also called condensates, likely play essential roles in maintaining normal retina function. However, currently, there is a critical gap in our knowledge: What proteins form higher-order assemblies under normal conditions within the retina and what function do these structures serve? Herein, we present data suggesting that protein aggregation is identifiable in multiple retinal layers of normal, healthy murine retina, and briefly discuss the potential contributions of aggregated proteins to normal retinal function, with a focus on the photoreceptor inner and outer segment.

MiR130b from Schlafen4+ MDSCs stimulates epithelial proliferation and correlates with preneoplastic changes prior to gastric cancer.

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4- cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b. RESULTS: MicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

Snow goose hepatitis B virus (SGHBV) envelope and capsid proteins independently contribute to the ability of SGHBV to package capsids containing single-stranded DNA in virions.

UNLABELLED: Hepadnaviruses selectively package capsids containing mature double-stranded DNA (dsDNA) genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing single-stranded DNA (ssDNA) in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA, with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the "capsid packaging signal" on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions than do those replicating DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis. IMPORTANCE: Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that "capsid maturation," and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication.

Compositional changes in the humin fraction resulting from the long-term cultivation of an Irish grassland soil: Evidence from FTIR and multi-NMR spectroscopies.

Soil humin (HN), a major long-term sink for carbon in the pedosphere, plays a key role in the global carbon cycle, and has been less extensively studied than the humic and fulvic acids components. There are increasing concerns about the depletions of soil organic matter (SOM) arising from modern soil cultivation practices but there has been little focus on how HN can be altered as the result. This study has compared the HN components in a soil under cultivation for wheat for >30 years with those from an adjacent contiguous soil that had been under long-term grass for all that time. A urea-fortified basic solution isolated additional humic fractions from soils that had been exhaustively extracted in basic media. Then further exhaustive extractions of the residual soil material with dimethyl sulfoxide, amended with sulphuric acid isolated what may be called the "true" HN fraction. The long-term cultivation resulted in a loss of 53 % soil organic carbon in the surface soil. Infrared and multi-NMR spectroscopies showed the "true" HN to be dominated by aliphatic hydrocarbons and carboxylated structures, but with clear evidence for lesser amounts of carbohydrate and peptide materials, and with weaker evidence for lignin-derived substances. These lesser-amount structures can be sorbed on the soil mineral colloid surfaces and/or covered by the hydrophobic HN component or entrained within these which have strong affinities for the mineral colloids. HN from the cultivated site contained less carbohydrate and more carboxyl groups suggesting slow transformations took place resulting from the cultivation, but these were much slower than for the other components of SOM. It is recommended that a study be made of the HN in a soil under long-term cultivation for which the SOM content has reached a steady state and where HN will be expected to dominate the components of SOM.

Isolation and fractionation of soil humin using alkaline urea and dimethylsulphoxide plus sulphuric acid.

Humin, the most recalcitrant and abundant organic fraction of soils and of sediments, is a significant contributor to the stable carbon pool in soils and is important for the global carbon budget. It has significant resistance to transformations by microorganisms. Based on the classical operational definition, humin can include any humic-type substance that is not soluble in water at any pH. We demonstrate in this study how sequential exhaustive extractions with 0.1 M sodium hydroxide (NaOH) + 6 M urea, followed by dimethylsulphoxide (DMSO) + 6% (v/v) sulphuric acid (H(2)SO(4)) solvent systems, can extract 70-80% of the residual materials remaining after prior exhaustive extractions in neutral and aqueous basic media. Solid-state (13)C NMR spectra have shown that the components isolated in the base + urea system were compositionally similar to the humic and fulvic acid fractions isolated at pH 12.6 in the aqueous media. The NMR spectra indicated that the major components isolated in the DMSO + H(2)SO(4) medium had aliphatic hydrocarbon associated with carboxyl functionalities and with lesser amounts of carbohydrate and peptide and minor amounts of lignin-derived components. The major components will have significant contributions from long-chain fatty acids, waxes, to cuticular materials. The isolates in the DMSO + H(2)SO(4) medium were compositionally similar to the organic components that resisted solvation and remained associated with the soil clays. It is concluded that the base + urea system released humic and fulvic acids held by hydrogen bonding or by entrapment within the humin matrix. The recalcitrant humin materials extracted in DMSO + H(2)SO(4) are largely biological molecules (from plants and the soil microbial population) that are likely to be protected from degradation by their hydrophobic moieties and by sorption on the soil clays. Thus, the major components of humin do not satisfy the classical definitions for humic substances which emphasise that these arise from microbial or chemical transformations in soils of the components of organic debris.

A two-year incubation study of transformations of crop residues into soil organic matter (SOM) and a procedure for the sequential isolation and the fractionation of components of SOM.

Maize (Zea mays) stover, with its natural 13C abundance, was incubated for two years in a gravelly brown earth sandy loam soil that had been under long term cultivation to wheat (Triticum aestivum) for more than 30 years. The relative abundances of 13C in the maize amendment allowed the contributions of the stover to be traced in the components of soil organic matter (SOM) isolated and fractionated using a sequential exhaustive extraction (SEE) process that gave 16 distinct fractions. These were caracterised using elemental, δ13C, FTIR, and 13C NMR analyses. Emphasis is placed on results for two years of incubation but to some extent data are compared with those for similar fractions taken after one year of incubation. Amounts of maize-derived organic carbon in the humic (HA) and fulvic (FA) isolates were more than twice those in the fractions after one year of incubation. The NMR results highlighted compositional differences between the fractions and showed increased contributions of lignin to the HAs and FAs (and especially in the cases of the HAs) as pH increased, and it was evident that humification was taking place after two years of incubation. The most recalcitrant humin fraction, isolated in the final solvent in the sequence, dimethylsulphoxide (DMSO) and sulfuric acid, is composed predominantly of methylene moieties, is compositionally and structurally very different from the humic and hydrophilic isolates, but identical to that which did not dissolve in the solvent. That suggests that exhaustively pre-extracting soil with the NaOH/urea solvent system used will allow a truly representative humin to be obtained using the DMSO/acid solvent system.

Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli.

For many proteins, aggregation is one part of a structural equilibrium that can occur. Balancing productive aggregation versus pathogenic aggregation that leads to toxicity is critical and known to involve adenosine triphosphate (ATP) dependent action of chaperones and disaggregases. Recently a second activity of ATP was identified, that of a hydrotrope which, independent of hydrolysis, was sufficient to solubilize aggregated proteins in vitro. This novel function of ATP was postulated to help regulate proteostasis in vivo. We tested this hypothesis on aggregates found in Xenopus oocyte nucleoli. Our results indicate that ATP has dual roles in the maintenance of protein solubility. We provide evidence of endogenous hydrotropic action of ATP but show that hydrotropic solubilization of nucleolar aggregates is preceded by a destabilizing event. Destabilization is accomplished through an energy dependent process, reliant upon ATP and one or more soluble nuclear factors, or by disruption of a co-aggregate like RNA.

Characterization of transformations of maize residues into soil organic matter.

An awareness of the transformation of plant residues returned to cultivated soils is vital for a better understanding of carbon cycles, the maintenance of soil fertility and the practice of a sustainable agriculture. The transformation of maize (Zea mays L) straw residues into soil organic matter (SOM) in a one year incubation experiment was studied in a soil that had been under long term cultivation with wheat (Triticum aestivum L) for >30years. A novel sequential exhaustive extraction and fractionation procedure isolated a series of fractions of SOM. The samples were characterized by elemental and δ13C analyses, by amino acids and neutral sugars analyses, by Fourier transformed infrared (FTIR) spectrometry, and by solid state 13C nuclear magnetic resonance (NMR) spectroscopy and with chemical shift anisotropy (CSA) -filter and dipolar dephasing (DD) spectral editing NMR techniques. The δ13C data indicated that 59% and 38% of the newly transformed organic carbon was in the humic and fulvic acid fractions, respectively, and in general a greater proportion of the transformed carbon was in the fractions isolated at the higher pH values. Results for SOM fractions from the amended soil indicate dominant contributions from carbohydrate and lignin-like material, and that can be clearly identified by FTIR, CP/TOSS, and spectral editing of CSA-filter and DD. The compositions of the fractions from the amended and non-amended soils fractions can be clearly differentiated using principal component analysis (PCA) for the data collected. The sequential extraction procedure showed that the hydrophilicity of humic fractions increased as the result of the maize amendment, and the aromaticity of the fraction decreased. The data may give some indications of transformations that take place during humification processes.

Mutagenic activities of biochars from pyrolysis.

Biochar production, from pyrolysis of lignocellulosic feedstocks, agricultural residues, and animal and poultry manures are emerging globally as novel industrial and commercial products. It is important to develop and to validate a series of suitable protocols for the ecological monitoring of the qualities and properties of biochars. The highly sensitive Salmonella mutagenicity assays (the Ames test) are used widely by the toxicology community and, via the rat liver extract (S9), can reflect the potential for mammalian metabolic activation. We examined the Ames test for analyses of the mutagenic activities of dimethylsulphoxide (DMSO) extracts of biochars using two bacterial models (S. typhimurium strains TA98 and TA100) in the presence and in the absence of the metabolic activation with the S9-mix. Tester strain TA98 was most sensitive in detecting mutagenic biochar products, and the contribution of S9 was established. Temperature and times of pyrolysis are important. Biochar pyrolysed at 400°C for 10min, from a lignocellulose precursor was mutagenic, but not when formed at 800°C for 60min, or at 600°C for 30min. Biochars from poultry litter, and manures of calves fed on grass had low mutagenicities. Biochar from pig manure had high mutagenicity; biochars from manures of cows fed on a grass plus cereals, those of calves fed on mother's milk, and biochars from solid industrial waste had intermediate mutagenicities. The methods outlined can indicate the need for further studies for screening and detection of the mutagenic residuals in a variety of biochar products.

Amyloids assemble as part of recognizable structures during oogenesis in Xenopus.

A hallmark of Alzheimer's, Huntington's and similar diseases is the assembly of proteins into amyloids rather than folding into their native state. There is an increasing appreciation that amyloids, under specific conditions, may be non-pathogenic. Here we show that amyloids form as a normal part of Xenopus oocyte development. Amyloids are detectable in the cytosol and the nucleus using an amyloid binding dye and antibodies that recognize amyloid structure. In the cytosol, yolk platelets are amyloid reactive, as are a number of yet to be characterized particles. In the nucleus, we find particles associated with transcription by RNA polymerase I, II and III and RNA processing contain amyloids. Nuclear amyloids remain intact for hours following isolation; however, RNase treatment rapidly disrupts nuclear amyloids.

Effects of Soluble Lignin on the Formic Acid-Catalyzed Formation of Furfural: A Case Study for the Upgrading of Hemicellulose.

A comprehensive study is presented on the conversion of hemicellulose sugars in liquors obtained from the fractionation of Miscanthus, spruce bark, sawdust, and hemp by using formic acid. Experimental tests with varying temperature (130-170 °C), formic acid concentration (10-80 wt%), carbohydrate concentrations, and lignin separation were carried out, and experimental data were compared with predictions obtained by reaction kinetics developed in a previous study. The conversions of xylose and arabinose into furfural were inherently affected by the presence of polymeric soluble lignin, decreasing the maximum furfural yields observed experimentally by up to 24%. These results were also confirmed in synthetic mixtures of pentoses with Miscanthus and commercial alkali lignin. This observation was attributed to side reactions involving intermediate stable sugar species reacting with solubilized lignin during the conversion of xylose into furfural.

Conversion of hemicellulose sugars catalyzed by formic acid: kinetics of the dehydration of D-xylose, L-arabinose, and D-glucose.

The pre-treatment of lignocellulosic biomass produces a liquid stream of hemicellulose-based sugars, which can be further converted to high-value chemicals. Formosolv pulping and the Milox process use formic acid as the fractionating agent, which can be used as the catalyst for the valorisation of hemicellulose sugars to platform chemicals. The objective of this study was to investigate the reaction kinetics of major components in the hemicelluloses fraction of biomass, that is, D-xylose, L-arabinose and D-glucose. The kinetics experiments for each sugar were performed at temperatures between 130 and 170 °C in various formic acid concentrations (10-64 wt %). The implications of these kinetic models on the selectivity of each sugar to the desired products are discussed. The models were used to predict the reaction kinetics of solutions that resemble the liquid stream obtained from the fractionation process of biomass using formic acid.

An integrated process for the production of platform chemicals and diesel miscible fuels by acid-catalyzed hydrolysis and downstream upgrading of the acid hydrolysis residues with thermal and catalytic pyrolysis.

This study evaluates an integrated process for the production of platform chemicals and diesel miscible biofuels. An energy crop (Miscanthus) was treated hydrothermally to produce levulinic acid (LA). Temperatures ranging between 150 and 200 °C, sulfuric acid concentrations 1-5 wt.% and treatment times 1-12 h were applied to give different combined severity factors. Temperatures of 175 and 200 °C and acid concentration of 5 wt.% were found to be necessary to achieve good yield (17 wt.%) and selectivities of LA while treatment time did not have an effect. The acid hydrolysis residues were characterized for their elemental, cellulose, hemicellulose and lignin contents, and then tested in a small-scale pyrolyzer using silica sand and a commercial ZSM-5 catalyst. Milder pretreatment yielded more oil (43 wt.%) and oil O(2) (37%) while harsher pretreatment and catalysis led to more coke production (up to 58 wt.%), less oil (12 wt.%) and less oil O(2) (18 wt.%).

Compositional changes in the hydrophobic acids fraction of drainage water from different land management practices.

Dissolved organic matter (DOM) can play a key role in many environmental processes, including carbon cycling, nutrient transport and the fates of contaminants and of agrochemicals. Hydrophobic acids (Ho), the major components of the DOM, were recovered from the drainage waters from well-drained (WDS) and poorly-drained (PDS) Irish grassland soils in lysimeters, amended with N fertiliser (F) and with bovine urine (U) and were studied using 1D and 2D solution-state Nuclear Magnetic Resonance (NMR) spectroscopy. The Diffusion Edited (DE) (1)H NMR spectra indicated that the Ho consisted largely of larger molecules, or of molecules that formed rigid aggregates, and the 1D and the 2D (Heteronuclear Multiple Quantum Coherence - HMQC, the Total Correlation Spectroscopy - TOCSY, and the Nuclear Overhauser Effect - NOESY) spectra indicated that the samples were composed of lignin residues, carbohydrates, protein/peptides, and aliphatic components derived from plant waxes/cuticular materials and from microbial lipids. The F amendments increased the concentrations of Ho in the waters by 1.5 and 2.5 times those in the controls in the cases of WDS and PDS, respectively. The lignin-derived components were increased by 50% and 300% in the cases of the Ho from the WDS and PDS, respectively. Applications of F + U decreased the losses of Ho, (compared to the F amendments alone) and very significantly decreased those of the lignin-derived materials, indicating that enhanced microbial activity from U gave rise to enhanced metabolism of the Ho components, and especially of lignin. In contrast the less biodegradable aliphatic components containing cuticular materials increased as the result of applications of F + U. This study helps our understanding of how management practices influence the movement of C between terrestrial and aquatic environments.

Unraveling the structural components of soil humin by use of solution-state nuclear magnetic resonance spectroscopy.

Humin is the most recalcitrant and least understood fraction of soil organic matter. By definition, humin is that fraction not extracted by traditional aqueous alkaline soil extractants. Here we show that > or = 70% of the traditional humin fraction is solubilized when 0.1 M NaOH + 6 M urea and dimethyl sulfoxide (DMSO) + 6% H2SO4 are used in series after conventional extraction. Multidimensional solution-state NMR is applied in this study to gain an understanding of the major constituents present in these "solubilized humin fractions". The spectra indicated strong contributions from five main categories of components, namely, peptides, aliphatic species, carbohydrates, peptidoglycan, and lignin. Diffusion edited spectroscopy indicated that all species are present as macromolecules (or stable aggregate species). Although the distribution of the components is generally similar, peptidoglycan is present at significant levels supporting a higher microbial contribution to humin than to humic and fulvic fractions. The abundance of plant- and microbial-derived materials found does not exclude "humic" materials (e.g., oxidized lignin) or the presence of novel compounds at lower concentrations but suggests that a large proportion of humin is formed from classes of known compounds and parent biopolymers.

Studies of the compositions of humic acids from Amazonian Dark Earth soils.

The compositions of humic acids (HAs) isolated from cultivated and forested "Terra Preta de Indio" or Amazonian Dark Earth soils (anthropogenic soils) were compared with those from adjacent non-anthropogenic soils (control soils) using elemental and thermogravimetric analyses, and a variety of solid-state nuclear magnetic resonance techniques. The thermogravimetric index, which indicates the molecular thermal resistance, was greater for the anthropogenic soils than for the control soils suggesting polycyclic aromatic components in the former. The cultivated anthropogenic soils were more enriched in C and depleted in H than the anthropogenic soils under forest, as the result of the selective degradation of aliphatic structures and the possible enrichment of H-deficient condensed aromatic structures. The combination of variable amplitude cross-polarization (VACP) and chemical shift anisotropy with total suppression of spinning sidebands experiments with composite pi pulses could be used to quantify the aromaticity of the HAs from the anthropogenic soils. From principal component analysis, using the VACP spectra, it was possible to separate the different constituents of the HAs, such as the carboxylated aromatic structures, from the anthropogenic soils and plant derived compounds. The data show that the HAs from anthropogenic soils have high contents of aryl and ionisable oxygenated functional groups, and the major functionalities from adjacent control soils are oxygenated functional groups from labile structures (carbohydrates, peptides, and with evidence for lignin structures). The anthropogenic soils HAs can be considered to be more recalcitrant, and with more stable reactive functional groups which may, in part, explain their more sustainable fertility due to the organic matter contribution to the soil cation exchange capacity.

Characterisation of black carbon-rich samples by (13)C solid-state nuclear magnetic resonance.

There are difficulties in quantifying and characterising the organic matter (OM) in soils that contain significant amounts of partially oxidised char or charcoal materials. The anthropogenic black carbon (BC), such as that found in the Terra Preta de Indio soils of the Amazon region, is a good example of the OM that is difficult to analyse in such soils. (13)C direct polarisation/magic angle spinning (DP/MAS) at high MAS frequency, (1)H-(13)C cross polarisation (CP)/MAS with total suppression of spinning sidebands (TOSS), and chemical shift anisotropy (CSA) filter nuclear magnetic resonance techniques have been applied successfully for quantifying the different components of OM. However, because pyrogenic materials present strong local magnetic susceptibility heterogeneities, the use of CSA-filter and TOSS make the pulse sequences very sensitive to imperfections in the pi pulses. In this study, the DP/MAS pulse sequence was replaced by a CP with a radio frequency ramp--variable amplitude CP (VACP)--VACP/MAS pulse sequence, and composite pi pulses were used in the CSA-filter and TOSS pulse sequences. In that way, the component functionalities in a humic acid from a BC soil were successfully determined. The spectrometer time needed was greatly decreased by employing this VACP/MAS technique. This development provides an accurate method for characterising BC-rich samples from different origins.

The application of LC-NMR and LC-SPE-NMR to compositional studies of natural organic matter.

Non-living natural organic matter (NOM) is ubiquitous in the oceans, atmosphere, sediments, and soils, and represents the most abundant organic carbon reserves on earth. However, a large proportion is considered to be "molecularly uncharacterized" because the inherent complexity of NOM is problematic when applying conventional analytical techniques. This manuscript presents initial applications of LC-NMR (1H) and LC-SPE-NMR (1H) to the studies of NOM isolated from water and soil. LC-NMR is applied to dissolved natural organic matter (DNOM) collected from freshwater environments, and both LC-NMR and LC-SPE-NMR are applied to an alkaline soil extract. The polar and complex nature of the DNOM samples limits conventional reversed phase separation, which can be partially overcome with the use of an ion pair reagent, although such an approach further complicates the NMR detection. LC-SPE-NMR of the soil alkaline extract was encouraging, and specific components in the mixture could be assigned. This work demonstrates that it is both possible to separate and concentrate specific components in NOM such that NMR detection is possible. As NMR information will be critical in unraveling the novel and/or complex structures in NOM this represents a key analytical hurdle in this area.

Molecular structures and associations of humic substances in the terrestrial environment.

Here we show, for the first time, evidence of the primary molecular structures in humic substances (HS), the most abundant naturally occurring organic molecules on Earth, and their associations as mixtures in terrestrial systems. Multi-dimensional nuclear magnetic resonance (NMR) experiments show us that the major molecular structural components in the mixtures operationally defined as HS are aliphatic acids, ethers, esters and alcohols; aromatic lignin derived fragments; polysaccharides and polypeptides. By means of diffusion ordered spectroscopy, distinct diffusion coefficients consistent with relatively low molecular weight molecules were observed for all the components in the mixtures, and saccharides were the largest single class of component present. Liquid chromatography NMR confirmed that HS components can be easily separated and nuclear Overhauser effect (NOE) enhancements support the finding that the components are of relatively low molecular weight