Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose

Propionibacterium acidipropionici was grown in a fed-batch culture, fed with glucose or lactate, or mixtures of lactate and glucose. Lactate and glucose were always simultaneously consumed. As co-substrate, glucose modified the propionate:acetate molar ratio (P/A) and increased the fraction of carbon used for biomass production. A P/A of 7.63 was obtained with a lactate:glucose molar ratio of 4; a P/A value of 1.34 was obtained with lactate alone and 1.85 with glucose alone. The fraction of carbon recovered in biomass was 0.09 for glucose, 0.12 for lactate, and 0.21 for a lactate:glucose molar ratio of 4.     

Lipid lowering and antioxidant effects of Amomum subulatum seeds (Family Zingiberaceae) in cholesterol fed rabbits

In the present study, hypolipidemic activity of fraction (50:50; CHCl₃:CH₃OH) of Amomum subulatum (Zingiberaceae) seeds was evaluated in cholesterol-fed rabbits. Hyperlipidemia induced by feeding atherogenic diet for 120 days resulted in a significant increase in serum total cholesterol, phospholipid and triglyceride levels when compared with control group. The levels of LDL and VLDL-cholesterol were increased significantly, but the HDL-cholesterol ratio was decreased. The changes in the antioxidant parameters were accompanied by an increase in lipid peroxidation and reduction in glutathione (GSH) and catalase activity. The level of lipid peroxidation was reduced whereas GSH content and catalase activity were elevated after the treatment with A. subulatum fraction at the dose level of 100 mg/kg.b.wt/day. A significant reduction was observed in total cholesterol, triglyceride, phospholipid, LDL and VLDL cholesterol where as HDL-cholesterol ratio was increased after administration of A. subulatum. The results of the present study indicate that fraction of A. subulatum possesses lipid-lowering and antioxidant activity and could be beneficial in the treatment of hyperlipidemia.     

Effects of Obesity and Weight Loss on Cardiac Function and Valvular Performance

KARASON, KRISTJAN, INGEMAR WALLENTIN, BO LARSSON, LARS SJOSTROM. Effects of obesity and weight loss on cardiac function and valvular performance. Obes Res. 1998;6:422–429. Objective : To study the consequences of long-standing obesity on myocardial function and valvular performance and to determine the effects of weight loss on these cardiovascular features. Research Methods and Procedures : We included 41 patients with obesity referred for weight-reducing gastroplasty, 31 patients with obesity who received dietary recommendations, and 43 lean subjects. Body weight and blood pressure were measured, and cardiac function and valvular performance were estimated echocardiographically. Left ventricular ejection fraction was used to assess systolic heart function, and the ratio of transmitral early to atrial (E/A) peak flow velocity was used as an estimate of diastolic filling. All three study groups were investigated at baseline, and the two groups with obesity were re-examined at 1-year follow-up. Results : Patients with obesity had higher blood pressure, greater cardiac output, lower ejection fraction, and reduced E/A ratio, compared with lean subjects (p<0.01). Surgical treatment of obesity led to significant decreases in body weight, whereas body weight remained unchanged in the group treated with dietary recommendations (p<0.001). In the weight loss group, blood pressure and cardiac output decreased and the E/A ratio increased (p<0.001). Left ventricular ejection fraction tended to increase in the weight loss group and decrease in the obese control group p<0.01). No significant valvular disease was observed in any of the subjects with obesity at baseline or after weight loss. Discussion : We conclude that weight reduction in subjects with obesity is associated with improvements in left ventricular diastolic filling and has favorable effects on left ventricular ejection fraction. Neither obesity nor weight loss seem to promote valvular heart disease.     

Characterization of Membrane-bound Spermidine Dehydrogenase of Citrobacter freundii

Spermidine dehydrogenase found in the membrane fraction of Citrohacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and γ-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.     

LM fibroblast plasma membrane subfractionation by affinity chromatography on Con A-sepharose

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-side-out orientation of the vesicles. Similarly the asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5′-nucleotidase activity, and (Na⁺ + K⁺)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and Arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.     

NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ∼3500 and ∼100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.     

Isolation and partial characterization of uronic acid-containing glycoproteins from Mucor rouxii

Five different fractions containing uronic acids associated with protein were isolated from the cytoplasm of the filamentous form of Mucor rouxii. A signle fraction was isolated from the cell wall by hot sodium dodecyl sulfate followed by ion exchange column chromatography. Two cytoplasmic entities (peaks I and II) were not adsorbed to DEAE Bio-Gel A. The molecular mass of peaks I to V ranged from 16.5 to 210 kDa. The protein-uronic acid ratios were different for each fraction. The cell wall fraction showed a molecular mass of 16.5 kDa, similar to that of peak II but with differences in chromatographic behavior and protein-uronic acid ratio. The possible role of these molecules as acceptors of sugar residues during polyuronide chain growth is discussed.     

Contribution of Disulfide Bonds to the Characteristics of Casein Micelles

Drainage in foam was analyzed by the capillary model proposed by Haas and Johnson. Foam was produced by introducing air into an ovalbumin solution through a spinneret. The electric conductivity at selected positions in the foam and the drained liquid height below the foam were measured at constant intervals. The measured electric conductivity was converted to the liquid volume fraction by Prager’s equation. The capillary model described well not only the liquid leakage rate from foam but also the liquid volume fraction in foam. The ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border was much larger than the value estimated from Haas and Johnson’s result. It was confirmed that a time constant, T, involved in the model was suitable for estimating foam stability only by one parameter. However, T is affected not only by material characteristics but also by the foam height. On the other hand, since the ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border reflects the material characteristics, it may be used for comparing the foam stability between materials with the same viscosity and density.     

Ketone components in the contact sex pheromone of the white-spotted longicorn beetle, Anoplophora malasiaca, and pheromonal activity of synthetic ketones

Two active fractions were found during the isolation of contact sex pheromone of female elytra of the white‐spotted longicorn beetle, Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae), in addition to fraction of hydrocarbons that had previously been identified. One fraction was essential to evoke a series of precopulatory behaviors of males toward a glass dummy when coated together with the hydrocarbon blend. The other fraction enhanced this activity when added to the mixture. From the latter synergistic fraction, we isolated five novel compounds and identified them as 10‐heptacosanone, (Z)‐18‐heptacosen‐10‐one, (18Z,21Z)‐heptacosa‐18,21‐dien‐10‐one, (18Z,21Z,24Z)‐heptacosa‐18,21,24‐trien‐10‐one, and 12‐heptacosanone by GC‐MS and NMR analyses. A blend of four of these synthetic ketones, without 12‐heptacosanone, in the ratio and concentration found in female elytra extract (250 : 400 : 1000 : 180 ng FE^?1) showed greater synergistic effect than the natural fraction containing the ketones. This effect was canceled out by further addition of 12‐heptacosanone (100 ng FE^?1), which was still comparable to the effect of the natural ketone fraction.     

The Influence of Liquid to Soil Ratios on Arsenic and Lead Bioaccessibility in Reference and Field Soil

In vitro bioaccessibility testing is gaining popularity as a tool to estimate the oral bioavailability of contaminants in soil for human health risk assessment (HHRA). Bioaccessibility tests are used to measure the bioaccessible fraction of a contaminant in soil, which can then be used to estimate the bioavailable fraction. Inherent uncertainties are associated with bioaccessibility tests. Various test parameters need to be carefully considered in their development, including the liquid to soil (L/S) ratio employed. We used L/S ratios (v:wt) ranging from 25 ml:1 g to 1,000 ml:1 g in a modified relative bioaccessibility extraction procedure to investigate the effects on bioaccessibility of lead and arsenic in field and reference soils. General trends of increased percent bioaccessibility of lead and arsenic with increasing L/S ratio were observed in the reference soil. A similar positive relationship was observed for lead in the field soil; soluble arsenic concentrations were below the detection limit and data were insufficient to observe a trend. Percent bioaccessibility was significantly affected at each extreme of the L/S ratios tested (p < .05). Biological relevance, technical feasibility, and mathematical relationships with in vivo results should be considered when selecting an appropriate L/S ratio for bioaccessibility testing.     

An Acyl Migration in Acetohalogenoglucosamines

Two triterpenoid alcohols, i.e., α- and β-amyrin have been isolated from the unsaponifiable fraction of the ethereal extract of Ilex latifolia Thunberg (Aquifoliaceae). The ratio of the amyrin contents, i.e., α-amyrin to β-amyrin was about 4:1. Stearic acid has also been isolated from the fatty-acid fraction of the hydrolysates.     

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of ATP in rat cerebral cortex

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4°C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A.70, 765–768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (Pl) fraction and cold-soluble tubulin in the supernatant (Sl) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of ³²P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of ³²P.     

Development of an improved procedure for isolation and purification of exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483

A method was developed for the isolation and purification of exopolysaccharide (EPS) produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 that can be adapted for industrial-scale operation. Hydrolyzed milk medium, which was ultrafiltered to remove molecular species larger than 2.5×10⁵ Da, was found to be a suitable growth medium for the bacteria, which produced approximately 400 mg EPS/l . Optimal isolation of EPS was achieved using centrifugation, filtration and ethanol precipitation methods. Insoluble and soluble EPS fractions were obtained. The soluble fraction was purified using a series of ethanol precipitations to achieve approximately 98% (w/w) purity. This fraction consisted of galactose, glucose, rhamnose and mannose in the ratio of approximately 5:1:0.6:0.5, with traces of glucosamine.     

Features of Grana Organization in Pea Chloroplasts

Two fractions of membrane fragments—the pellets precipitated at 1300 and 20000 g (fractions G1.3 and G20, respectively)—were isolated from pea (Pisum sativum L.) chloroplasts after solubilization with digitonin. These fragments assigned to grana displayed the following differences: (1) in spectra of low-temperature fluorescence, the ratio of short-wave and long-wave band intensities, as well as integrated intensity of the whole spectrum, were higher for G1.3 than for G20 fraction; (2) in excitation spectra of long-wave fluorescence, the ratio of peaks at 650 and 680 nm and integrated intensity of the spectrum were higher for G1.3 than for G20 fraction; and (3) the shapes of fluorescence excitation spectra differed for G1.3 and G20. These results indicate that the two fractions examined differed in proportion of photosystem I and photosystem II complexes, as well as in organization of these complexes. The size of light-harvesting antenna was larger in PSI complexes of G1.3 fraction, owing, in particular, to a higher content of chlorophyll a/b-protein complexes in this fraction. After repeated digitonin fragmentation of G1.3 and G20 preparations, more than 80% of G1.3 fraction was decomposed into lighter fragments, whereas G20 fraction was resistant to fragmentation (it lost about 10% of its material). Analysis of the data suggests the presence of two structurally different types of thylakoids in grana. The yield of G20 fraction (about 20%) is comparable to the ratio between the number of intergranal thylakoids, connected to granum in pea chloroplasts, and the total number of thylakoids in this granum. Based on these data, we assume that G20 fraction represent the fragments of intergranal thylakoids that extend into the granum.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 499–506.Original Russian Text Copyright © 2005 by Kochubei, Shevchenko, Bondarenko.     

Enzymic feruloylation of arabinoxylan-trisaccharide by feruloyl-CoA:arabinoxylan-trisaccharide O-hydroxycinnamoyl transferase from Oryza sativa

Feruloyl-CoA:arabinoxylan-trisaccharide O-hydroxycinnamoyl transferase, which catalyzes the transfer of ferulic acid from Fer-CoA to arabinoxylan-trisaccharide in the formation of feruloyl arabinoxylan-trisaccharide (Fer-AXX), has been found in an ionically bound fraction and a cytosol fraction of suspension-cultured rice (Oriza sativa L. cv. Nipponbare) cells. Analysis of reaction products by high-performance liquid chromatography showed the formation of product A, which is one of the transfer products having the same retention time as authentic Fer-AXX. Product A was purified by reverse-phase chromatographies to characterize its structure. The isolated product A showed the same ultraviolet spectrum and molecular weight on fast atom bombardment mass spectrometric analysis as those of authentic Fer-AXX. Alakaline saponification of product A released ferulic acid and oligosaccharide. The released oligosaccharide consisted of arabinose and xylose in a molar ratio of 1:2. These results support the identity of product A as feruloylated arabinoxylan-trisaccharide and show the existence of a feruloyltransferase catalyzing the feruloylation of a hemicellulosic fragment. Received: 14 July 2000 / Accepted: 22 August 2000     

Soluble Leptin Receptor and Soluble Receptor‐Bound Fraction of Leptin in the Metabolic Syndrome

Objective: In obesity, plasma leptin is high and soluble leptin receptor (sOb‐R) levels are low, resulting in a low fraction of bound leptin. The aim of this study was to investigate the influence of insulin resistance (IR) and the metabolic syndrome (MS) on sOb‐R concentration and the bound‐free ratio of leptin. Research Methods and Procedures: sOb‐R, leptin levels, and homeostasis model assessment (HOMA) index for IR were determined in 76 middle‐aged obese or overweight men. Results: Concentration of sOb‐R and soluble receptor‐bound fraction of leptin were lowest in the highest tertile of HOMA‐IR. sOb‐R and the bound‐free ratio of leptin correlated with HOMA‐IR, leptin concentration, and waist‐to‐hip ratio independently of age, BMI, and fat mass. Leptin and waist‐to‐hip ratio were the sole independent determinants of sOb‐R concentration, and BMI, HOMA‐IR, and visceral adipose tissue were independent determinants of the bound fractin of leptin. sOb‐R concentration and the bound fraction of leptin decreased with increasing numbers of components of the MS, resulting in lower sOb‐R concentration and a lower fraction of bound leptin in men with the MS. Discussion: IR and abdominal obesity are associated with low sOb‐R concentration and low bound‐free ratio of leptin independent of fat mass. Low sOb‐R concentration and low bound‐free ratio of leptin segregate with components of the MS. We suggest that low sOb‐R levels and a low fraction of specifically bound leptin are markers of leptin resistance, which is independently associated with IR and abdominal obesity and may constitute an additional component of the MS.     

Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation

Abstract : The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P₃ fraction, but cytoplasmic ACh contained in the S₃ fraction was not significantly affected. The P₃ fraction is known to be heterogeneous ; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K⁺-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [³H]ACh from [³H]choline, the ratio of [³H]ACh in the S₃ to P₃ fractions was not much changed by exposure of tissue to calyculin A or okadaic acid ; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.     

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.     

DEGRADATION OF THE CELL-WALL POLYSACCHARIDE OF PORPHYRIDIUM SP. (RHODOPHYTA) BY MEANS OF ENZYMATIC ACTIVITY OF ITS PREDATOR,GYMNODINIUM SP. (PYRROPHYTA)1

The dinoflagellate Gymnodinium sp., which preys specifically on cells of the red microalga Porphyridium sp., possesses enzymes that degrade exocellular polysaccharides of the Porphyridium sp. A crude extract of Gymnodinium sp. was applied to this polysaccharide, and the degradation products were characterized by charge and size separations. Charge separation revealed the presence of a fraction that was not found in the native polysaccharide. This fraction, which was eluted from an anion-exchange resin with water alone, was composed mostly of glucose and xylose (in a 1:1 weight ratio). Size separation of the degradation products revealed three fractions; the molecular weight of the main one was 5 × 10⁶ daltons, whereas that of the native polysaccharide was 7 × 10⁶ daltons. The carbohydrate composition of these fractions was determined. Although the main product of degradation had a relatively high molecular weight, its viscosity was significantly reduced relative to the native polysaccharide. Additional enzymatic degradation is required for further exploration of the structure of the exocellular polymer of Porphyridium sp.