Maximal release of highly bifidogenic soluble dietary fibers from industrial potato pulp by minimal enzymatic treatment

Potato pulp is a poorly utilized, high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp mainly consists of the tuber plant cell wall material and is particularly rich in pectin, notably galactan branched rhamnogalacturonan I type pectin which has previously been shown to exhibit promising properties as dietary fiber. The objective of this study was to solubilize dietary fibers from potato pulp by a one-step minimal treatment procedure and evaluate the prebiotic potential of the fibers. Statistically designed experiments were conducted to investigate the influence of enzyme type, dosage, substrate level, incubation time, and temperature on the enzyme catalyzed solubilization to define the optimal minimal enzyme treatment for maximal fiber solubilization. The result was a method that within 1 min released 75% [weight/weight (w/w)] dry matter from 1% (w/w) potato pulp treated with 1.0% (w/w) [enzyme/substrate (E/S)] pectin lyase from Aspergillus nidulans and 1.0% (w/w) E/S polygalacturonase from Aspergillus aculeatus at pH 6.0 and 60 °C. Molecular size fractionation of the solubilized fibers revealed two major fractions: one fraction rich in galacturonic acid of 10–100 kDa indicating mainly homogalacturonan, and a fraction >100 kDa rich in galactose, presumably mainly made up of β-1,4-galactan chains of rhamnogalacturonan I. When fermented in vitro by microbial communities derived from fecal samples from three healthy human volunteers, both of the solubilized fiber fractions were more bifidogenic than fructo-oligosaccharides (FOS). Notably the fibers having molecular masses of >100 kDa selectively increased the densities of Bifidobacterium spp. and Lactobacillus spp. 2–3 times more than FOS.     

Effects of the Dry–Wet Hydrological Shift on Dissolved Organic Carbon Dynamics and Fate Across Stream–Riparian Interface in a Mediterranean Catchment

Abstract The stream–riparian interface, characterized by a dynamic and complex hydrology, is an important control point for nutrient fluxes and processing between terrestrial and aquatic systems. Predicted alterations in the discharge regime in Mediterranean climate regions make it necessary to understand the effects of abrupt hydrological transition between dry and wet conditions on the transport and fate of dissolved organic carbon (DOC) across the stream–riparian interface. In this study, the concentrations and fate of total DOC (TDOC) and a subset of four molecular weight fractions (<1 kDa, 1–10 kDa, 10–100 kDa, >100 kDa) were investigated in stream water and riparian groundwater during autumn of 2003 and 2004. The two study periods were characterized by contrasting antecedent hydrological conditions: the streamflow was interrupted in summer 2003 but was permanent in summer 2004. Comparison of the two study periods indicates that an abrupt dry–wet hydrological transition amplifies the water exchange across the stream–riparian interface and favors retention of up to 57% of the TDOC that flows across the interface. Furthermore, the efficiency of DOC retention across the stream-riparian interface also varies greatly depending on DOC molecular size. More than 70% of DOC fractions higher than 10 kDa were retained, whereas the smaller fraction (less than 1 kDa) was nearly conserved. Consequently, our study helps to clarify the effects of extreme hydrological events on DOC transport in running waters in Mediterranean regions.     

Fluorescence and amino acid characteristics of molecular size fractions of DOM in the waters of Lake Biwa

Dissolved organic matter (DOM) in the waters from Lake Biwa, Japan was fractionated using tangential flow ultrafiltration, and subsequently characterized by fluorescence properties and amino acids. While major dissolved organic carbon (DOC), UV absorbance (Abs), humic-like fluorescence (Flu) and total hydrolyzed amino acids (THAA) occurred in the less than 5 kDa molecular size fraction, they were not evenly distributed among various molecular size fractions. Flu/Abs ratios increased, and THAA/DOC ratios decreased with decreasing molecular size. Humic-like fluorescence occurred in all molecular size fractions, but protein-like fluorescence only occurred in the 0.1 m-GF/F fraction. Subtle differences in amino acid compositions (both individuals and functional groups) were observed between various molecular size fractions, this may indicate the occurrence of DOM degradation from higher to lower molecular weight. The results reported here have significance for further understanding the sources and nature of DOM in aquatic environments.     

<Emphasis Type="Italic">Vibrio cholerae</Emphasis> in waters of the Sunderban mangrove: relationship with biogeochemical parameters and chitin in seston size fractions

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 μM) and nitrogen (9.1 ± 3.3 μM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1–2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with ~98% of the total in the fraction <20 μm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes >20 μm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes <20 μm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5–10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant (>22 μM N and 100 μM Si). However, nitrite increased ~9 times in the outer sector, reaching ~1.2 μM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality.     

Abnormal Gangliosides are Localized in Lipid Rafts in Sanfilippo (MPS3a) Mouse Brain

Allogenic stem cell transplantation can reduce lysosomal storage of heparan sulfate-derived oligosaccharides by up to 27 % in Sanfilippo MPS3a brain, but does not reduce the abnormal storage of sialolactosylceramide (G_M3) or improve neurological symptoms, suggesting that ganglioside storage is in a non-lysosomal compartment. To investigate this further we isolated the Triton X100-insoluble at 4 °C, lipid raft (LR) fraction from a sucrose-density gradient from cerebral hemispheres of a 7 month old mouse model of Sanfilippo MPS3a and age-matched control mouse brain. HPLC/MS/MS analysis revealed the expected enrichment of normal complex gangliosides, ceramides, galatosylceramides and sphingomyelin enrichment in this LR fraction. The abnormal HS-derived oligosaccharide storage material was in the Triton X100-soluble at 4 °C fractions (8–12),whereas both GM3 and sialo[GalNAc]lactosylceramide (GM2) were found exclusively in the LR fraction (fractions 3 and 4) and were >90 % C18:0 fatty acid, suggesting a neuronal origin. Further analysis also revealed a >threefold increase in the late-endosome marker bis (monoacylglycerol) phosphate (>70 % as C22:6/22:6-BMP) in non-LR fractions 8–12 whereas different forms of the proposed BMP precursor, phosphatidylglycerol (PG) were in both LR and non-LR fractions and were less elevated in MPS3a brain. Thus heparan sulfate-derived oligosaccharide storage is associated with abnormal lipid accumulation in both lysosomal (BMP) and non-lysosomal (GM3 and GM2) compartments.     

13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

Evidence of protein-tyrosine kinase activity in Catharanthus roseus roots transformed by Agrobacterium rhizogenes

Homogenate fractions (soluble and particulate) from transformed roots of Catharanthus roseus (L.) G. Don showed several phosphorylated proteins when incubated with γ-[³²P]ATP. The phosphorylation in the proteins of 55, 40, 25, 18 and 10 kDa in the particulate fraction and 63 kDa in the soluble fraction was resistant to alkali treatment. Several proteins in both fractions gave a positive signal with monoclonal antiphosphotyrosine antibodies. In-situ phosphorylation in both fractions showed several proteins that cross-reacted with the antiphosphotyrosine antibodies. Tyrosine kinase activity was detected using an exogenous substrate RR-SRC, a synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60^src. This activity was inhibited by genistein, a tyrosine kinase inhibitor. These results indicate, for the first time, the presence of protein-tyrosine kinase (EC 2.7.1.112) activity in transformed plant tissues. Received: 29 March 1997 / Accepted: 21 May 1997     

Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica

In this study, three alginate fractions with different molecular weights and ratios of mannuronic acid (M) to guluronic acid (G) were prepared by enzymatic hydrolysis and ultrafiltration to assess the antioxidant property of alginates from Laminaria japonica with molecular weight below 10 kDa. The antioxidant properties of different molecular weight alginates were evaluated by determining the scavenging abilities on superoxide, hydroxyl, and hypochlorous acid and inhibitory effect on Fe²⁺-induced lipid peroxidation in yolk homogenate. The results showed that low molecular weight alginates exhibited high scavenging capacities on superoxide, hydroxyl, and hypochlorous acid radicals and good inhibition of Fe²⁺-induced lipid peroxidation in yolk. By comparison, alginate A1 with molecular weight below 1 kDa and M/G of 1.84 had better scavenging activity on superoxide, hydroxyl, and hypochlorous acid radicals in vitro than A2 (1–6 kDa), A3 (6–10 kDa), ascorbic acid, and carnosine. With similar M/G ratio, A2 exhibited better antioxidant activity on superoxide and hypochlorous acid radicals than A3. However, fraction A3 with molecular weight of 6–10 kDa exhibited higher inhibitory ability on lipid peroxidation in yolk in vitro than A1 and A2. The results indicated that molecular weight played a more important role than M/G ratio on alginate to determine the antioxidant ability. By comparison, low molecular weight alginates composed of guluronic acid and mannuronic acid exhibited better antioxidant ability on oxygen free radicals than sulfated polysaccharides from L. japonica in our previous study and represent a good source of marine polysaccharide with potential application as natural antioxidant.     

Nitrogen deposition effects on soil organic matter chemistry are linked to variation in enzymes,ecosystems and size fractions

Recent research has dramatically advanced our understanding of soil organic matter chemistry and the role of N in some organic matter transformations, but the effects of N deposition on soil C dynamics remain difficult to anticipate. We examined soil organic matter chemistry and enzyme kinetics in three size fractions (>250 μm, 63–250 μm, and <63 μm) following 6 years of simulated atmospheric N deposition in two ecosystems with contrasting litter biochemistry (sugar maple, Acer saccharum—basswood, Tilia americana and black oak, Quercus velutina—white oak, Q. alba). Ambient and simulated (80-kg NO₃ ⁻–N ha⁻¹ year⁻¹) atmospheric N deposition were studied in three replicate stands in each ecosystem. We found striking, ecosystem-specific effects of N deposition on soil organic matter chemistry using pyrolysis gas chromatography/mass spectrometry. First, furfural, the dominant pyrolysis product of polysaccharides, was significantly decreased by simulated N deposition in the sugar maple–basswood ecosystem (15.9 vs. 5.0%) but was increased by N deposition in the black oak–white oak ecosystem (8.8 vs. 24.0%). Second, simulated atmospheric N deposition increased the ratio of total lignin derivatives to total polysaccharides in the >250 μm fraction of the sugar maple–basswood ecosystem from 0.9 to 3.3 but there were no changes in other size classes or in the black oak–white oak ecosystem. Third, simulated N deposition increased the ratio of lignin derivatives to N-bearing compounds in the 63–250 and >250 μm fractions in both ecosystems but not in the <63 μm fraction. Relationships between enzyme kinetics and organic matter chemistry were strongest in the particulate fractions (>63 μm) where there were multiple correlations between oxidative enzyme activities and concentrations of lignin derivatives and between glycanolytic enzyme activities and concentrations of carbohydrates. Within silt-clay fractions (<63 μm), these enzyme-substrate correlations were attenuated by interactions with particle surfaces. Our results demonstrate that variation in enzyme activity resulting from atmospheric N deposition is directly linked to changes in soil organic matter chemistry, particularly those that occur within coarse soil size fractions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphorus speciation in Myall Lake sediment, NSW, Australia

The amount of phosphorus and its fractions in the sediment of Lake Myall, NSW, Australia, was assessed using a sequential extraction technique. Five sedimentary phosphorus reservoirs were measured, namely loosely sorbed phosphorus (NH₄Cl–P), iron associated phosphorus (BD–P), calcium bound phosphorus (HCl–P), metal oxide bound phosphorus (NaOH–P) and residual phosphorus (organic and refractory P, Res-P). Samples were taken from the deep and shallow sites of the lake. During the analysis, the average concentrations of each fraction of phosphorus was calculated. The results depicted that the total phosphorus (TP) content and chemically extractable phosphorus in both fine and coarse sediment fractions from the deep sites of the lake were significantly higher than those of the shallow sites, except for HCl–P extracted from the fine sediment fraction. Sediment TP was also strongly and positively correlated to sediment Fe. The phosphorus in the sediment mainly consisted of BD–P and Res-P, while NH₄Cl–P and HCl–P only contributed a minor part. The rank order of the different phosphorus extracts was similar for the two sites, namely Residual-P > BD–P > NaOH–P > HCl–P > NH₄Cl–P.     

Purification and characterization of mycoferritin from Fusarium verticillioides MRC 826

The fungus Fusarium verticillioides MRC 826 (ascomycetes species), a toxigenic isolate is capable of synthesizing mycoferritin only upon induction with iron in yeast extract sucrose medium. The molecular mass, yield, iron and carbohydrate contents of the purified mycoferritin were 460 kDa, 0.010 mg/g of wet mycelia, 1.0 and 40.2%, respectively. Native gel electrophoresis of the mycoferritin revealed two bands possibly representing isoforms of ferritin. Subunit analysis by SDS–PAGE showed a single protein subunit of ~24 kDa suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy revealed an apparent particle size of 100 Å. N-terminal amino acid sequencing of mycoferritin showed identities with other eukaryotic ferritin sequences. The spectral characteristics were similar to equine spleen ferritin. However, circular dichroic spectra revealed a higher degree of helicity. Functionally, induction of mycoferritin minimizes the pro-oxidant role of iron.     

A comparison of phytoplankton size-fractions in Mondsee,an alpine lake in Austria: distribution,pigment composition and primary production rates

Production rates, abundance, chlorophyll a (Chl a) concentrations and pigment composition were measured for three size classes (<2 μm, 2–11 μm and >11 μm) of phytoplankton from May to December 2000 in deep, mesotrophic, alpine lake Mondsee in Austria. The study focuses on differences among phytoplankton size fractions characterised by their surface area to volume ratio ([mm² l⁻¹: mm³l⁻¹]), pigment distribution patterns and photosynthetic rates. Particular attention was paid to autotrophic picophytoplankton (APP, fraction <2 μm) since this size fraction differed significantly from the two larger size fractions. Among the three fractions, APP showed the highest surface area to volume ratios and a high persistence in the pattern of lipophilic pigments between temporarily and spatially successive samples (about 80% similarity of pigment composition between samples over seasons and depths). The epilimnetic abundance of APP varied seasonally with an annual maximum of 180 × 10³ cells ml⁻¹ in June (at 4–9 m). The minimum (October at 12 m) was more than an order of magnitude lower (4.9 × 10³ ml⁻¹). APP peaked during autumn and contributed between 24% and 42% to the total area-integrated Chl a (10–23 mg m⁻²) and between 16% and 58% to total area-integrated production (5–64 mg m⁻²  h⁻¹) throughout seasons.     

Optimizing the biodegradation of two keratinous wastes through a Bacillus subtilis recombinant strain using a response surface methodology

Statistical optimization of the biodegradation of two keratinous wastes directed by Bacillus subtilis recombinant cells was carried out by means of a response surface methodology. A Box–Behnken design was employed to predict the optimal levels of three variables namely, keratin percent, incubation time and inoculum size. Analysis of variance revealed that, only keratin percent had the highest significant effect. Canonical analysis and ridge max analysis were used to get the optimal levels of the three predictors along with the optimum levels of the responses. The optimal sets of predicted and validated levels of the three variables were [7.69% (w/v) feathers, 96.58 h and 1.28% (v/v) inoculum size] and [8% (w/v) feathers, 98.45 h, 3.9% (v/v) inoculum size] to achieve the highest levels of soluble proteins (1.25–1.7 mg/ml) and NH₂-free amino groups (245.82–270.0 μmol leucine/ml), respectively upon using three optimized feathers-based media. These values represented 83.67–100% and 100% adequacy for the models of soluble proteins and NH₂-free amino groups, respectively. While, [8.23% (w/v) sheep wool, 5.52% (v/v) inoculum size and 46.58 h] and [8.33% (w/v) sheep wool, 5.89% (v/v) inoculum size and 63.46 h] were the optimal sets of predicted and validated levels of the above variables to achieve the highest yields of soluble proteins (3.4–4.6 mg/ml) and NH₂-free amino groups (290.9–302.0 μmol leucine/ml), respectively upon using three optimized sheep wool-based media. These values represented 100% adequacy for the models of soluble proteins and NH₂-free amino groups. By the end of the optimization strategy, a fold enhancement (2.14–2.43 and 1.78–2.12) in the levels of released soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized feathers-based media. However, a fold enhancement (4.25–5.75 and 2.42–2.5) in the levels of soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized sheep wool-based media. Data would encourage pilot scale optimization of the biodegradation of these wastes.     

Bacterial Consumption of Humic and Non-Humic Low and High Molecular Weight DOM and the Effect of Solar Irradiation on the Turnover of Labile DOM in the Southern Ocean

The decomposition of dissolved organic matter (DOM) in pelagic ecosystems is mediated primarily by heterotrophic bacteria, but transformation by short-wave solar radiation may play an important role in surface waters, in particular when humic substances constitute a substantial fraction of the DOM pool. Most of the studies examining bacterial decomposition and photochemical transformation of DOM stem from limnetic and coastal marine systems and much less information is available from oceanic environments. To examine the bacterial decomposition of humic and non-humic DOM in the Southern Ocean we carried out microcosm experiments in which we measured bacterial growth on isolated fractions of humic and non-humic DOM of the size classes <3 kDa and >3 kDa. Experiments carried out at the Polar Front showed a preferential bacterial growth on non-humic DOM and in particular on the size fraction <3 kDa. Bacterial growth, measured as bacterial biomass production, on non-humic DOM accounted for 74% to 88% of the total growth on all four DOM fractions. In experiments in the Antarctic circumpolar current and the coastal current under pack ice, bacterial growth was 6× lower than at the Polar Front, and humic and non-humic DOM was consumed to equal amounts. The size fraction <3 kDa was always preferred. Experiments examining the effect of solar radiation on the release of dissolved amino acids (DAA) and carbohydrates (DCHO) and their subsequent bacterial utilization showed a stimulating effect on glucose uptake and the release of DAA at the Polar Front but an inhibition in the eastern Weddell Sea. Ultraviolet-B was the most effective component of the solar radiation spectrum tested. Effects of UV-B on glucose uptake and release of DAA were positively correlated with concentrations of humic-bound DAA. The data imply that at low concentrations, e.g., <100 nM (amino acid equivalent), UV-irradiation reduces, whereas at concentrations >100 nM UV-irradiation stimulates glucose uptake and release of DAA as compared to dark conditions.     

Distribution and characteristics of molecular size fractions of freshwater-dissolved organic matter in watershed environments: its implication to degradation

Distributions of molecular size and fluorescence properties of dissolved organic matter (DOM) in the Lake Biwa and Lake Baikal watersheds were investigated using the cross-flow ultrafiltration technique and three-dimensional fluorescence measurements. From the fluorescence properties, protein-like substances were usually found in the 0.1 μm-GF/F fraction (the Durapore membrane retentate of the GF/F filtrate) of the lake DOM. The results indicated autochthonous production of protein-like organic-matters in the lake environment. Fulvic acid (FA)-like components were composed of two fractions with respect to fluorescence properties and molecular size. Two FA-like fluorescence peaks, which showed different fluorescence peak positions in the excitation-emission matrix (EEM), were partly fractionated by the molecular size of 5000 daltons (5 kDa). The FA-like fluorescence peak position of the <5-kDa fraction was observed at the shorter wavelength region compared with that of the fraction between 5 kDa and 0.1 μm (5 kDa20.1 μm fraction). A blue shift of the FA-like fluorescence peak position as well as a decrease in the molecular size of the DOM was observed in lake samples. The relative contribution of the <5 kDa fraction to the DOC concentration was high in lake waters (68%–79%) compared with river waters (44%–68%), suggesting characteristic changes in molecular size between riverine and lacustrine DOM. DOM of the 5 kDa–0.1 μm fraction was relatively higher in river waters than in lake waters. These findings coincided with in situ distributions of the fluorescence properties and molecular size of DOM found in both stream and lake environments. These results indicate that FA-like substances from forested watersheds are decomposed qualitatively and quantitatively in the river-lake environment by photochemical and biological processes.     

Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger

The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360–440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 °C. The fraction containing the enzyme activity contained at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0. Received: 9 May 1999 / Accepted: 30 November 1999     

Airborne Poaceae pollen in Porto (Portugal) and allergenic profiles of several grass pollen types

This work aims to investigate the presence of airborne grass pollen and to identify antigenic and allergenic profiles from eight different grass species collected in the Porto region (Portugal). Poaceae airborne pollen, sampled using a Hirst-type volumetric trap during 2003–2007, was the second most abundant type, and high concentrations were found from April to August. Pollen proteins extracted from the eight grass species collected were separated by SDS-PAGE, being the allergenic profile investigated by immunoblotting using sera from atopic patients and maize profilin polyclonal antibody (ZmPRO3). Pollen extract profiles showed several bands ranging from 10 to 97 kDa. In immunoblotting studies, a low molecular weight protein (12–13 kDa) was recognized by profilin antibody. Also, in all pollen extracts except Zea mays, the IgE binding proteins of 12–13 kDa were detected in sera from the 25 patients with different sensitization profiles presenting high IgE values (>80 kU/l). This protein can be considered as a potential causal agent of the allergic respiratory diseases.     

Arecoline and the 30–100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified ≥10 kDa fraction (ANE ≥ 10 K) and subsequently to the 30–100 kDa fraction (ANE 30–100 K). ANE and ANE ≥10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE ≥ 10 K, and ANE 30–100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30–100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser²⁴⁴⁸. In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser²⁴⁴⁸ phosphorylation are capable of triggering apoptosis and autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Shyun-Yeu Liu, Mei-Huei Lin, Yu-Rung Hsu and Ya-Yun Shih contributed equally to this work.     

Lead induced antioxidant defense system in pigeon pea and its impact on yield and quality of seeds

Toxic effects of lead (>0.2 mM Pb) were measured in pigeon pea (Cajanus cajan Mill) cv. UPAS grown in sand culture as reduction in growth, yield, and quality of seeds. Leaves containing >38 μg g⁻¹ Pb showed oxidative damage as decrease in chlorophyll content and induction of antioxidants such as carotenoids, proline, and non-protein thiol contents with enhanced activities of SOD and peroxidase. At excess (>0.2 mM) Pb, accumulation of >1,000 μg Pb g⁻¹ root tissue was associated with increase in non-protein thiol content. It is concluded that inhibition in root-to-shoot translocation of Pb and induction in the level of proline, chloroplast pigments, and non-protein thiols and activities of antioxidant enzymes SOD and peroxidase at <0.2 mM Pb could have protected the pigeon pea plants from the deleterious effects of Pb. However, excess Pb at >0.2 mM showed a decline in yield, boldness, and quality of seeds despite the expression of an additional band each of Cu–Zn SOD and peroxidase isoform. The threshold of toxicity and toxicity values in pigeon pea leaves of plants exhibiting 10 and 33% yield depression at 27 days after metal supply were 27 and 56 μg Pb g⁻¹, respectively.