HMW Fraction Research Articles

Purification of Polyhydroxybutyrate Synthase from Its Native Organism, Ralstonia eutropha: Implications for the Initiation and Elongation of Polymer Formation in Vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.

Textural and Bile Acid‐Binding Properties of Muffins Impacted by Oat β‐Glucan with Different Molecular Weights

ABSTRACTWater‐soluble β‐glucan (BG) extracted from a high‐BG oat line was treated with different amounts of lichenase (1→3)(1→4)‐β‐d‐glucanase) enzyme to yield three different molecular weight (MW) BG extracts. Low (LMW‐BG, 157,000), medium (MMW‐BG, 277,000), and high (HMW‐BG, 560,000) MW BG extracts were added to plain muffin formulations at a level of 0.52% (0.42% in the batter, 0.52% in the resultant muffins) to investigate the effect of MW of BG on textural and bile acid (BA) binding properties of the muffins. In addition, treatments were prepared containing LMW‐BG, MMW‐BG, and HMW‐BG extracts in amounts providing equivalent batter firmness as determined on a texture analyzer. Resultant BG concentrations (and per serving amounts) of these muffins were 1.36% (0.81 g/60 g muffin), 1.05% (0.63 g/60 g muffin), and 0.52% (0.31 g/60 g muffin), respectively; thus, the LMW treatment complied with a U.S. Food and Drug Administration health claim requiring 0.75 g of BG per serving. The firmness, springiness, and BA‐binding capacity of the muffins were unaffected by the MW of BG. However, when added at the maximum limit for equivalent batter firmness, the LMW treatment was more firm and less springy than the HMW treatment. Furthermore, BA‐binding capacities of LMW and MMW fractions tended to be greater than that of the HMW fraction when added at the maximum limit. These results add further evidence to the importance of fine‐tuning BG structure to provide maximum health benefits while maintaining high product quality.

Bacterial utilization of size-fractionated dissolved organic matter

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 64:299-309 (2011) - DOI: https://doi.org/10.3354/ame01529 Bacterial utilization of size-fractionated dissolved organic matter Vishwas B. Khodse, Narayan B. Bhosle* Marine Corrosion and Material Research Division, National Institute of Oceanography (CSIR), Dona Paula, 403004, Goa, India *Email: bhosle@nio.org ABSTRACT: Dissolved organic matter (DOM) is an important source of organic carbon for sus­taining the growth of heterotrophic bacteria. We investigated the bacterial utilization of high-­molecular-weight (HMW; >30 kDa to 0.22 µm) and low-molecular-weight (LMW; >10 to 30 kDa) fractions of DOM. DOM was collected from a station in Dona Paula Bay, on the west coast of India, and fractionated into HMW and LMW portions. Each size fraction was inoculated with a natural population of bacteria and incubated over a period of 15 d at room temperature (28 ± 2°C); during this period, sub-samples were removed and the following were measured: dissolved organic carbon (DOC), total dissolved carbohydrate (TDCHO), free monosaccharide (MCHO), dissolved uronic acid (DURA), δ13C, bacterial abundance (BA), and bacterial production (BP). The LMW fraction was isotopically heavier (δ13C = −23.7 to −21.7‰) than the HMW fraction (δ13C = −27.0 to −26.2‰), and the initial TDCHO content of these fractions was 25 and 16%, respectively. The initial DURA content was similar in the LMW DOM (7.4% DOC) and the HMW DOM (7.0% DOC). BA and BP were consistently higher in the LMW DOM than in the HMW DOM. In 15 d incubation, greater proportions of DOC (35%), TDCHO (76%) and DURA (36%) were utilized in the LMW DOM than in the HMW DOM. This suggests that the LMW DOM was more biologically reactive, i.e. it was utilized more rapidly, than the HMW DOM. It appears that the bioreactivity and origin of the DOM strongly influenced its utilization by natural heterotrophic bacteria. KEY WORDS: Dissolved organic matter · Size fractions · Carbohydrates · Polysaccharides · Bacteria · Bacterial production · Dona Paula Bay Full text in pdf format PreviousCite this article as: Khodse VB, Bhosle NB (2011) Bacterial utilization of size-fractionated dissolved organic matter. Aquat Microb Ecol 64:299-309. https://doi.org/10.3354/ame01529 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 64, No. 3. Online publication date: September 20, 2011 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2011 Inter-Research.

Comparative Flavan-3-ol Profile and Antioxidant Capacity of Roasted Peanut, Hazelnut, and Almond Skins

In the present study, the flavan-3-ol composition and antioxidant capacity of roasted skins obtained from the industrial processing of three commonly consumed tree nuts (i.e., peanuts, hazelnuts, and almonds), as well as fractions containing low and high molecular weight (LMW and HMW) flavan-3-ols, were studied with the aim of assessing their potential as a source of flavonoids. Roasted peanut and hazelnut skins presented similar total phenolic contents, much higher than that of almond skins, but their flavan-3-ol profiles, as determined by LC-ESI-MS and MALDI-TOF MS, differed considerably. Peanut skins were low in monomeric flavan-3-ols (19%) in comparison to hazelnut (90%) and almond (89%) skins. On the other hand, polymeric flavan-3-ols in peanut and almond skins occurred as both A- and B-type proanthocyanidins, but in peanuts the A forms (up to DP12) were predominant, whereas in almonds the B forms (up to DP8) were more abundant. In contrast, hazelnuts were mainly constituted by B-type proanthocyanidins (up to DP9). The antioxidant capacity as determined by various methods (i.e., total antioxidant capacity, ORAC, DPPH test, and reducing power) was higher for whole extracts from roasted hazelnut and peanut skins than for almond skins; however, the antioxidant capacities of the HMW fraction of the three types of nut skins were equivalent despite their different compositions and DPs. Nevertheless, the large variation in flavan-3-ol concentration, structural composition, type of interflavan linkage, and DP found among the three types of nut skins suggests large difference in their expected in vivo biological activities.

Protein profile and sensorial properties of rye breads

The protein profile of four types of traditional rye breads formed on rye flours with the extraction rate of 100, 95, 85 and 70% and baked at 260 °C for 40 min was investigated as a nutritional quality indicator. A fractionation process was applied to discriminate high-molecular- (HMW >3 kDa) and low-molecular weight compounds (LMW 3 kDa) and filtrates (LMW <3 kDa). The dependence between sensory quality of rye breads and composition on soluble proteins, available lysine, carbohydrates and total phenolic compounds was provided. Baking caused a decrease in rye bread protein content. The content of available lysine was the lowest in crusts and it was dependent on the rye flour extraction rate taken for rye bread making. The SDS-PAGE analysis showed that the primary structure of the rye bread proteins was modified in the highest extent in the crust’s bread of all types. The analyses of both retentates and filtrates after ultrafiltration demonstrated that the most of carbohydrates and phenolic compounds were incorporated into the HMW fraction suggesting their conjugation with proteins due to the Maillard reaction. Compounds able to react with Folin Ciocalteu reagent were also detected in filtrates of crust’s extracts (MW ≤3 kDa) from all breads indicating their distribution between LMW and HMW fractions. About 22.5, 27, 11.2 and 46.8% of the phenol compounds forming crusts of breads based on the flours with extraction rates of 100, 95, 85 and 70% were recovered in the corresponding LMW fractions.The sensory analysis of the four types of rye breads indicated for a high correlation between overall quality of breads and their content of carbohydrates. The highest correlation was noted for a rye bread formed on flour with extraction rate of 95%. This finding suggested that the content of carbohydrates as a substrate for Maillard reaction has had an influence on palatability by consumers of rye bread formed on flour with the extraction rate of 95% which had also the highest overall quality.

Structure and Water Relations of Melanoidins Investigated by Thermal, Rheological, and Microscopic Analysis

Water soluble melanoidins (WSM) from a glucose-glycine model system were obtained and separated into high (HMW) and low (LMW) molecular weight fractions. Adsorption isotherm and GAB and BET fitting parameters of WSM were determined. Physical and structural properties were investigated through thermal and rheological analysis. The LMW fraction exhibited thermal properties similar to that of oligosaccharides and the HMW fraction was compatible with a complex and large molecular size biopolymer structure, while both exhibited gel like properties. Intrinsic viscosity values were consistent with spherical hydrodynamic volumes.

In this issue: Proteomics – Clinical Applications 5/2009

AbstractIn this issue of Proteomics – Clinical Applications you will find the following highlighted articles:Proteomics gets savory: salivating againLast month in Proteomics 09/09 we looked at pea aphid saliva, this month we're considering human saliva. Vitorino et al. came out with all guns blazing as they hunted down the sources of the peptides found in human whole saliva (WS). Peptides in WS from six subjects were isolated and concentrated by three methods. They were then centrifuged to remove bacteria and debris with (i) nothing, (ii) 4 M guanidine‐HCl, or (iii) 0.1% trifluoroacetic acid added. Separation into HMW and LMW fractions was done by filtration through 100‐, 50‐, and 10 kDa cut‐off filters. Size limits were 700 to 16 000 Da. Samples were further analyzed by gelatinase zymogram electrophoresis, LC‐MALDI‐TOF/TOF, MALDI‐MS, LC‐MS/MS, and ex vivo proteolysis. Results: 2294 peptides identified (2210 LMW, of those 76% debris or plasma), 78 common to all samples. Cathepsin D appears to have been responsible for &gt;40% of protease activity.Vitorino, R. et al., Proteomics Clin. Appl. 2009, 3, 528–540.Hepatocellular carcinoma markers: highest of the high, lowest of the lowHepatocellular carcinoma (HCC) rears its ugly head more than half a million times a year. It stands fifth on the frequency list but third on the killer list. If recognized in time, it can be controlled surgically. Thus the call for better biomarkers. Liver markers face the problem of being mostly serum proteins and must compete with highly abundant serum albumin and immunoglobulins. Wang et al. took a different approach, looking at both the secretomes and transcriptomes of HCC and normal tissues in serum‐free culture. Combining a five‐fold over‐expressed HCC marker with a five‐fold under‐expressed organ marker gave results better than either marker alone. The ratio of the pair, CHI3L1 and MASP2, had an area under curve score of 0.97, separately the AUC scores were 0.92 and 0.83 respectively.Wang, J. et al., Proteomics Clin. Appl. 2009, 3, 541–551.Multiple melanoma markers from plasmaMelanoma is one of the least frequent but most fatal skin cancers with a mortality rate of &gt;80% and growing worldwide. Patients and research would benefit from improved prognostic and diagnostic markers, so Takikawa et al. took up the challenge. After depleting late‐stage melanotic and normal plasmas of the six most abundant protein species, samples were subjected to two dimensions of chromatography and, finally, MALDI‐TOF. After analysis and subtracting overlaps, 24 myeloma specific proteins remained, after applying stricter selection criteria, the count was down to nine. Four of the nine proteins also showed elevated transcript levels. Expression was also validated by Western blots and immunohistochemistry. One marker, PPBP, also shows a potential for being a prognostic indicator as well as a therapeutic target.Takikawa, M. et al., Proteomics Clin. Appl. 2009, 3, 552–562.

Cover Picture: Proteomics – Clinical Applications 5/2009

AbstractIn this issue of Proteomics – Clinical Applications you will find the following highlighted articles:Proteomics gets savory: salivating againLast month in Proteomics 09/09 we looked at pea aphid saliva, this month we're considering human saliva. Vitorino et al. came out with all guns blazing as they hunted down the sources of the peptides found in human whole saliva (WS). Peptides in WS from six subjects were isolated and concentrated by three methods. They were then centrifuged to remove bacteria and debris with (i) nothing, (ii) 4 M guanidine‐HCl, or (iii) 0.1% trifluoroacetic acid added. Separation into HMW and LMW fractions was done by filtration through 100‐, 50‐, and 10 kDa cut‐off filters. Size limits were 700 to 16 000 Da. Samples were further analyzed by gelatinase zymogram electrophoresis, LC‐MALDI‐TOF/TOF, MALDI‐MS, LC‐MS/MS, and ex vivo proteolysis. Results: 2294 peptides identified (2210 LMW, of those 76% debris or plasma), 78 common to all samples. Cathepsin D appears to have been responsible for &gt;40% of protease activity.Vitorino, R. et al., Proteomics Clin. Appl. 2009, 3, 528–540.Hepatocellular carcinoma markers: highest of the high, lowest of the lowHepatocellular carcinoma (HCC) rears its ugly head more than half a million times a year. It stands fifth on the frequency list but third on the killer list. If recognized in time, it can be controlled surgically. Thus the call for better biomarkers. Liver markers face the problem of being mostly serum proteins and must compete with highly abundant serum albumin and immunoglobulins. Wang et al. took a different approach, looking at both the secretomes and transcriptomes of HCC and normal tissues in serum‐free culture. Combining a five‐fold over‐expressed HCC marker with a five‐fold under‐expressed organ marker gave results better than either marker alone. The ratio of the pair, CHI3L1 and MASP2, had an area under curve score of 0.97, separately the AUC scores were 0.92 and 0.83 respectively.Wang, J. et al., Proteomics Clin. Appl. 2009, 3, 541–551.Multiple melanoma markers from plasmaMelanoma is one of the least frequent but most fatal skin cancers with a mortality rate of &gt;80% and growing worldwide. Patients and research would benefit from improved prognostic and diagnostic markers, so Takikawa et al. took up the challenge. After depleting late‐stage melanotic and normal plasmas of the six most abundant protein species, samples were subjected to two dimensions of chromatography and, finally, MALDI‐TOF. After analysis and subtracting overlaps, 24 myeloma specific proteins remained, after applying stricter selection criteria, the count was down to nine. Four of the nine proteins also showed elevated transcript levels. Expression was also validated by Western blots and immunohistochemistry. One marker, PPBP, also shows a potential for being a prognostic indicator as well as a therapeutic target.Takikawa, M. et al., Proteomics Clin. Appl. 2009, 3, 552–562.

Natural organic matter from catchment to drinking water: a case study of Pori waterworks, Finland

In this study Natural organic matter (NOM) characteristics and variations of catchment samples (brooks and collector lakes) from Western Finland, and drinking water produced from the same catchment were examined. Seasonal and spatial NOM variations were followed by means of DOC and HPLC-SEC with UV and fluorescence detection. NOM decreased from drains to lakes by 35 to 75% and from drains to drinking water by 73 to 94%. Drains had a higher NOM content in summer and a lower NOM content in winter and spring. Lakes showed inverse patterns and had a higher NOM content in winter and spring and a lower NOM content in summer. HPLC-SEC separated 8 molecular weight fractions. In drains the HMW fractions represented up to 80% of the NOM, in lake waters HMW fractions accounted for 50 to 70% of the NOM. In drinking water IMW fractions dominated. Increased NOM in raw water during winter was associated with increased IMW fractions and the appearance of HMW fractions in drinking water, DOC increasing from 1.4 mg C/L in summer to 5.8 mg C/L in winter. SPH-Tryptophan correlated with the dissolved organic nitrogen and DOC of the samples. The drain affected by agriculture generally presented higher SPH-Tryptophan values than the unaffected drain.

Study on the effects of nitrilotriproprionic acid and 4,5-dihydroxy-1,3-benzene disulphonate on the fractionation of beryllium in human serum using graphite furnace atomic absorption spectrometry

BackgroundOccupational exposure to beryllium may cause Chronic Beryllium Disease (CBD), a lung disorder initiated by an electrostatic interaction with the MHC class II human leukocyte antigen (HLA). Molecular studies have found a significant correlation between the electrostatic potential at the HLA-DP surface and disease susceptibility. CBD can therefore be treated by chelation therapy. In this work, we studied the effect of two complexing agents, nitrilotriproprionic acid (NTP) and 4,5-dihydroxy-1,3-benzene disulphonate (Tiron), on the fractionation of beryllium in human serum analysed by graphite furnace atomic absorption spectrometry (GFAAS).ResultsWe found the average serum beryllium concentration of fourteen non-exposed individuals to be 0.53 (± 0.14) μg l-1, with 21 (± 3)% of the beryllium mass bound to the low molecular weight fraction (LMW), and 79 (± 3)% bound to the high molecular weight fraction (HMW). The addition of Tiron increased the beryllium mass in the HMW fraction, while NTP was not seen to have any influence on the fractionation of beryllium between the two fractions. NTP was, however, shown to complex 94.5% of the Be mass in the LMW fraction. The beryllium GFAAS detection limit, calculated as three times the standard deviation of 10 replicates of the lowest standard (0.05 μg L-1), was 6.0 (± 0.2) ng L-1.ConclusionThe concentration of beryllium or its fractionation in human serum was not affected by sex or smoking habit. On average, three quarters of the beryllium in serum were found in the HMW fraction. Of the two ligands tested, only Tiron was effective in mobilising beryllium under physiological conditions, thus increasing the Be content in the HMW fraction.

Molecular Size Affects Urine Excretion of Pentosan Polysulfate

In human subjects only a small percent of oral PPS is found in urine. Commercially available PPS is a heterogeneous mixture with varying molecular weights. Our hypothesis was that only the low molecular weight fraction reaches the urine. Urine was obtained from patients with IC who were chronically receiving PPS. The amount and molecular size of PPS in the urine were determined by enzyme-linked immunosorbent assay and molecular sieve chromatography. PPS was purified from Elmiron capsules and fractionated into LMW and HMW fractions. Urine recovery of PPS was measured in rabbits after oral or intravenous administration of unfractionated, LMW or HMW PPS. The median urine PPS level in 34 patients with IC was 1.2 microg/ml (range 0.5 to 27.7). All PPS recovered from IC urine was LMW. After intravenous administration in rabbits the median recovery in urine was 47.2% (range 19.7% to 73.2%) for unfractionated PPS, 74.6% (range 31.4% to 96.3%) for LMW and 3.3% (range 2.5% to 5.0%) for HMW. After oral administration in rabbits the median recovery in urine was 7.4% (range 2.1% to 46.0%) for LMW and 0.10% (range 0.0% to 0.3%) for HMW. In patients with IC who are on oral PPS the PPS recovered in the urine is all of LMW. In rabbits the HMW fraction of PPS is recovered in small amounts from urine after intravenous administration and not at all after oral administration.

Terrigenous organic matter sources and reactivity in the North Atlantic Ocean and a comparison to the Arctic and Pacific oceans

Lignin phenol concentrations and compositions in suspended particulate and dissolved organic matter (POM and DOM) were determined in samples from several locations and depths in the western North Atlantic Ocean. POM lignin phenol concentrations were ∼2-fold lower than previous measurements in the Pacific Ocean, but represented up to 28% of the total lignin phenols in the water column. Carbon-normalized yields of lignin phenols and δ 13C measurements of POM indicate that up to half of the suspended material in deep water is of terrigenous origin and appears to enter the ocean via atmospheric deposition. This finding emphasizes the need for future research into aerosol compositions and fluxes into the oceans. In contrast to POM, DOM lignin phenol concentrations were ∼2-fold higher in the Atlantic, with more than two thirds in the high-molecular weight (HMW; > 1 kDa) fraction. Elevated concentrations of dissolved lignin phenols in North Atlantic Deep Water (NADW) indicated that 10–16% of annual fluxes of terrigenous DOM from Arctic rivers could be entrained during NADW formation. The percentage of lignin phenols in the HMW fraction decreases progressively from the Arctic to Atlantic to Pacific Oceans, reflecting an increasing diagenesis of terrigenous DOM. The average residence time of lignin phenols in the Atlantic is ∼ 35 years. Terrigenous DOC accounts for 1–2% of the DOC in surface and deep waters of the North Atlantic.

Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses

The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves ( Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson’s r ranging from −0.63 to −0.78). On the other hand, theoretical maximum shell lengths ( L ∞) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently and most strongly correlated with the population variables (Pearson’s r ranging from −0.58 to −0.80). Our findings demonstrate, however, the difficulty of currently assigning to sub-cellular metal partitioning measurements (mainly Cd bound to the HMW fraction) any predictive role for population health, notably because of the influence of ecological confounding variables (e.g., the cumulative number of degree-days in the littoral zone, as is the case here). Metal contamination of our lakes has decreased markedly in the past 10 years and consequently we believe that the toxic effects of metals may have been replaced by some natural factors as the main agent for structuring the clam populations in these lakes.

Decrease in molecular weight of photosynthetic products of marine phytoplankton during early diagenesis

Changes in the size and neutral aldose (NAld) composition of phytoplankton photosynthetic products during the early diagenetic process were experimentally examined using 13C as a tracer. Most (94.7%) of the photosynthetically produced (P−) organic carbon (OC) was found to be particulate organic carbon (POC) after a 12‐h light incubation. An increase in the concentration of P‐dissolved organic carbon (DOC) was found after the shift to the dark incubation, probably due to a leakage of cellular constituents. An increase in the concentration of P‐DOC was mainly due to an increase in the high molecular weight (HMW: ≫10 kDa) fraction that reached its maximum concentration on day 3 and then declined at a relatively high rate. The change in the concentration of the low molecular weight (LMW: ≪10 kDa) DOC fraction was less marked than that in the HMW fraction, and the rate of decrease was much slower, indicating a more biorefractory nature of P‐LMW DOC. P‐dissolved carbohydrates (DCHO) accounted for 38% and 50% in P‐HMW DOC and P‐LMW DOC, respectively, after a 12‐h light incubation. The concentration of P‐LMW DCHO showed a rapid decrease in the early stage of the dark incubation, whereas the concomitant increase in the concentration of P‐LMW dissolved noncarbohydrate (DnonCHO) was noticed. The decrease in the contribution of CHO was noticed both in P‐POC and P‐HMW DOC fractions, but the decline rates were slower than that in P‐LMW DOC fraction. On day 60, the remaining P‐OC accounted for 4.6% of photosynthetic material originally produced. The distribution of the size fractions of the remaining P‐OC on day 60 (POC, 14%; HMW DOC, 22%; LMW DOC, 64%) indicates that phytoplankton photosynthetic products were rapidly degraded to the less bioreactive LMW DOM during early diagenesis. The present results indicate that the annual global ocean production rate of semilabile DOC with lifetimes exceeding 2 months is 1.91 PgC yr−1.

Genotoxicity of melanoidin fractions derived from a standard glucose/glycine model.

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.

Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient

The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure→bioaccumulation→effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels ( Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu catalyses the production of reactive oxygen species, to which the organism reacts by increasing activities of the two enzymes, thus preventing the accumulation of reactive oxygen species. Lipid concentrations in the gonad did not decrease with any of the measured toxicological parameters, suggesting that energy reserves for reproduction were not compromised in the metal-contaminated mussels. The results of the present study, where chronically exposed bivalves were collected from their natural habitat along a metal contamination gradient, contrast markedly with what would have been predicted on the basis of experimental metal exposures, and clearly demonstrate the need to study metal exposure→bioaccumulation→effects relationships in natural populations.

Determination of the molecular weight distribution of non-enzymatic browning products formed by roasting of glucose and glycine and studies on their effects on NADPH-cytochrome c-reductase and glutathione-S-transferase in Caco-2 cells.

After thermal treatment of a mixture of glucose and glycine for 2 h at 125 degrees C, about 60% of the starting material was converted into non-soluble, black pigments, whereas 40% of the mixture was still water-soluble. Dialysis of the latter fraction revealed 30.4% of low molecular weight compounds (LMWs; MW < 10,000 Da) and 10.0% high-molecular weight products (HMWs; MW > or = 10,000 Da). The water-soluble Maillard reaction products (MRPs) were separated by gel permeation chromatography and ultrafiltration, revealing that 60% of the water-soluble products of the total carbohydrate/amino acid mixture had MWs < 1,000 Da and consisted mainly of non-coloured reaction products. MRPs with MWs between 1,000 and 30,000 Da were found in comparatively low yields (about 1.3%). In contrast, about 31.1% of the MRPs exhibited MWs > 30,000 Da, amongst which 14.5% showed MWs > 100,000 Da, thus indicating an oligomerisation of LMWs to melanoidins under roasting conditions. To investigate the physiological effects of these MRPs, xenobiotic enzyme activities were analysed in intestinal Caco-2 cells. For Phase-I NADPH-cytochrome c-reductase, the activity in the presence of the LMW and HMW fraction was decreased by 13% and 22%, respectively. Phase-II glutathione-S-transferase activity decreased by 15% and 18%, respectively, after incubation with the LMW and the HMW fractions. Considering the different yields, 30% and 10%, respectively, of the LMW and the HMW fractions, the total amount of the LMW fraction present in the glucose-glycine mixture is more active in modulating these enzyme activities than that of the HMW fraction.

Size fractionation upon adsorption of fulvic acid on goethite: equilibrium and kinetic studies

We examined adsorption equilibrium and kinetics of an aquatic fulvic acid (XAD-8 resin extract) onto goethite (α-FeOOH). Molecular weight distributions were determined using high-pressure size exclusion chromatography (HPSEC). Overall adsorption isotherms and those of the most abundant intermediate molecular weight (IMW) fraction (1250–3750 Da) fit the Langmuir adsorption equation, as is commonly observed for humic substances. However, this overall fit masked the non-Langmuir isotherm shape of high and low molecular weight (HMW, LMW, respectively) fractions. We observed preferential adsorption of HMW fractions at low pH and of IMW fractions at higher pH. We also observed preferential adsorption of components with higher absorbance normalized to moles C (ε 280), probably reflecting greater aromaticity. Over the first 6 h of adsorption experiments, adsorbed organic carbon increased and weight average molecular weight ( M w ) of the organic matter remaining in solution decreased, consistent with slower adsorption of higher molecular weight components. Observations of fractionation upon adsorption agreed well with a field study showing lower M w and lower ε 280 organic matter in deeper ground water relative to surface and shallow ground water.

Molecular characterization of estuarine bacterial communities that use high- and low-molecular weight fractions of dissolved organic carbon

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 25:127-139 (2001) - doi:10.3354/ame025127 Molecular characterization of estuarine bacterial communities that use high- and low-molecular weight fractions of dissolved organic carbon Joseph S. Covert, Mary Ann Moran* Department of Marine Sciences, University of Georgia, Athens, Georgia 30602-3636, USA *Corresponding author. E-mail: mmoran@arches.uga.edu ABSTRACT: The composition of bacterial communities growing at the expense of high-molecular weight (HMW; >1000 Da) and low-molecular weight (LMW; <1000 Da) fractions of dissolved organic carbon from a southeastern US estuary was determined by sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene amplicons in 2 enrichment studies. 16S rDNA sequence analysis indicated that the bacterial communities growing on the LMW fraction were dominated by γ- and ε-Proteobacteria related to Pseudomonas fluorescens and Arcobacter nitrofigilis (accounting for 90% of the clones) while the communities using the HMW fraction were dominated by α-, b-, and γ-Proteobacteria and Cytophaga-Flexibacter-Bacteroides related to Rhizobium-Agrobacterium, Janthinobacterium lividum, Pseudomonas fluorescens, Marinobacterium georgiense, Pseudoalteromonas, and Sphingobacterium comitans (accounting for 98% of the clones). Methylotrophic bacteria were present in the inoculum community but not found in either LMW or HMW enrichments. T-RFLP analysis of the enrichment communities showed measurable changes in community composition during the enrichment period, and companion respiration assays confirmed utilization of sufficient HMW and LMW carbon to support several bacterial generations. Although the composition of the estuarine inoculum used for the 2 enrichment studies (conducted in April 1997 and May 1999) was quite similar, the communities developing on the HMW and LMW fractions differed between experiments, potentially reflecting temporal variations in the chemical composition of the dissolved organic carbon. KEY WORDS: Dissolved organic carbon (DOC) · Ultrafiltration · Terminal restriction fragment length polymorphism (T-RFLP) analysis · Bacterial community composition · 16S rDNA Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 25, No. 2. Online publication date: September 04, 2001 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2001 Inter-Research.

Environmental effects on the quality of two wheat genotypes: 1. quantitative and qualitative variation of storage proteins

A new method of sequential extraction of proteins followed by quantitative and qualitative determination by reverse-phased high-performance liquid chromatography (RP-HPLC) was used to analyse the effect of environmental conditions (3N fertilisation rates, two varieties, two sites and two growing seasons) on quantitative and qualitative variation of wheat storage proteins. The results showed that N supply (fertilisation and site) was the most important environmental factor affecting protein content and composition. The most important effect was quantitative: the total protein, protein unit and subunit contents increased with the supply of nitrogen to the grain. As grain protein increased, the gliadin and glutenin contents and the gliadin to glutenin ratio increased. Gliadin showed a higher correlation with total protein content than glutenin. In the variety Rinconada, glutenin content was higher than that in Bancal. As total glutenin increased, both high and low-molecular weight (HMW and LMW) fractions increased and their ratio (HMW/LMW) did not change significantly, despite a slope of LMW subunits two times greater (0.69) than that of HMW fractions (0.31). The quantity of each HMW subunit (RP-HPLC peak) increased with total HMW-glutenin, but their relative percentage increased for some peaks and decreased for others. The composition of the HMW subunit was more stable in Bancal than in Rinconada. This general pattern of variation was also characteristic of the LMW subunit. Concerning gliadin composition we noted that the content of different gliadin monomers (RP-HPLC peaks) or pools of monomers (α-, β-, γ-, ω-gliadin) increased with total gliadin content. The proportion of certain peaks was stable whereas the contribution of other peaks was related to the variation in gliadin content and depended on the variety.