The fractional precipitation of soil humic acid by ammonium sulphate

Summary A number of fractions have been obtained from a soil humic acid by salting-out and by leaching with ammonium sulphate. The fractions obtained are characterised by infra-red and ultra-violet spectroscopy and osmometry. The fractions removed at relatively low saturation with ammonium sulphate are less highly charged and contain more aliphatic hydrocarbon groups per unit weight than the fractions precipitated at high concentrations of salt. In addition, the number average molecular weight of successive fractions precipitated with ammonium sulphate decreases significantly.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Isolation and fractionation of hemicelluloses by graded ethanol precipitation from Caragana korshinskii

Caragana korshinskii hemicelluloses were isolated with 10% KOH at 25 °C for 10 h from the delignified materials. The alkali-extractable hemicelluloses were then successively sub-fractionated by graded precipitation at final ethanol concentrations of 10%, 20%, 30%, 45%, 60%, and 80%, respectively. Neutral sugars and molecular weight analyses of the six hemicellulosic subfractions revealed that the molecular weights and the distribution of branches along the xylan backbone are different among the hemicellulosic fractions obtained in various ethanol concentrations. The less branched hemicelluloses with large molecules were precipitated in lower ethanol percentages, while with the increasing ethanol concentrations, more branched hemicelluloses with low molecular weights were obtained. ¹H and ¹³C NMR studies revealed that the hemicellulosic subfraction precipitated at an ethanol concentration of 45% had a backbone of d-xylose residues and were branched mainly through 4-O-methyl-α-d-glucopyranosyl units.     

The fractionation of diphtheria toxoid by means of ammonium sulphate

At high toxoid concentrations as well as at low pH values the curves of the salting out process of diphtheria toxoid with ammonium sulphate have the classical S shape. At lower toxoid concentrations and at a pH value a little below 7 or above it, a part of the curve is horizontal or in some cases even descends. This suggests the presence of two flocculating components, one of which (component A) is precipitated at low (NH₄)₂SO₄ concentrations; this component is probably not immunogenic, or only poorly so. Fractionation and gel diffusion experiments confirmed the presence of these two components. In certain fractions of the toxoids the components may manifest themselves separately by the presence of two flocculation zones.For the purpose of an adequate purification of crude diphtheria toxoid by means of salting out, it appears advisable to keep the toxoid concentration below circa 1500 Lf/ml and the pH at a value higher than 6.5.     

Partial characterization and antimicrobial activity of peptides from <Emphasis Type="Italic">Amburana cearensis</Emphasis> seeds against phytopathogenic fungi and yeasts

The aim of this work was to study the antifungal action of a protein and peptide-rich fraction from Amburana cearensis seeds. Initially, proteins were extracted from seed flour in phosphate buffer and re-extracted with 1 M lithium chloride. The products obtained from both extractions were precipitated with ammonium sulfate at 70% saturation, and the precipitates were re-suspended in distilled water, heated at 80°C for 15 min and clarified by centrifugation at 12,000×g. The obtained fractions from both extractions were dialyzed, recovered by lyophilization and visualized in Tris–Tricine/SDS gel electrophoresis. The fractions obtained were rich in proteins of low molecular weight and were submitted to antifungal assays. Fraction S4 (extraction with phosphate buffer) inhibited the fungi, Colletotrichum lindemuthianum, Fusarium oxysporum, Fusarium solani, Candida albicans and Saccharomyces cerevisiae. Fractions P2 (re-extraction with lithium chloride) had a low or non-inhibitory effect on the fungi tested.     

Biological significance of the enantiomeric purity of drugs

The knowledge that enantiomers of chiral compounds may differ widely in biological activity, qualitatively as well as quantitatively, is not new. Nevertheless most of the pharmacological data available to date on chiral drugs are obtained from experiments with racemates which assume that the biological activity generally resides in one of the enantiomers. With the advancements made in stereospecific synthesis and stereoselective analysis of drugs pharmacologists are now offered new possibilities to explore the steric aspects of drug action. This survey will discuss pharmacological data obtained with enantiomer pairs of phenylethylamine derivatives which interact with adrenergic mechanisms. The degree of resolution is seldom specified in published work on stereoselectivity of drugs. In a recent study from our laboratory the enantiomers of the β₂-adrenoceptor agonist formoterol and their diastereomers have been evaluated. We found that the (R;R)-enantiomer was by far the most potent. However, the relative potencies obtained for the (R;S)-, (S;R), and (S;S)- isomers were critically dependent on the degree of enantiomeric purity. It is concluded that the certainty of potency ratios observed for chiral drugs is limited by the enantiomeric purity and by unspecific effects of the least active enantiomer at very high concentrations. © 1993 Wiley-Liss, Inc.     

A study of air particulate pollution in colombo using a nuclear-related analytical technique

Air particulate matter of two size fractions (coarse [2.5–10 Μm] and fine [less than 2.5 Μm]) were collected at an urban residential site (Colombo University ground) over a period of 12 mo during 1996 using a Gent PM10 stacked filter unit. Seventy-five sets of samples collected during this period were analyzed for 10 elements: Al, Si, K, S, Ca, Ti, Fe, Zn, Br, and Pb by energy-dispersive X-ray fluorescence, which is a multielement analysis. This is a highly sensitive technique enabling quantitative analysis of very low masses. The average concentrations of lead, sulfur, and bromine, which are generally associated with combustion products of automotive exhausts, dominate the fine fraction in this study. The minimum and maximum concentration of lead resulting in an annual average of 0.09 Μg/m³ was found to be 0.0042 and 0.441 Μg/m³ in particulate matter with less than 10 Μm equivalent aerodynamic diameter. The maximum concentration determined is well below the limits set by the World Health Organization (0.5–1.0 Μg/m³). The recommended value for Pb in Sri Lanka is 0.5 Μg/m³. Al, Si, Ca, Ti, and Fe exhibited enhanced concentrations in the coarse fraction, which probably originate from disturbed soil as a result of wind and traffic.     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Effect of a New Variety of Apis mellifera Propolis onMutans Streptococci

The effects of a new variety of propolis, from Northeastern Brazil (BA), on growth of mutans streptococci, cell adherence, and water-insoluble glucan (WIG) synthesis were evaluated. Propolis from Southeastern (MG) and Southern (RS) Brazil were also tested as an extension of our previous work. Ethanolic extracts of propolis (EEP) were prepared and analyzed by reversed-phase HPLC. For the antibacterial activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEPs against Streptococcus mutans, S. sobrinus, and S. cricetus were determined. Cell adherence of S. mutans and S. sobrinus to a glass surface was measured spectrophotometrically at 550 nm. WIG synthesized from sucrose by glucosyltransferase (Gtf) was extracted and quantified by the phenol-sulfuric method. The HPLC profile of the new variety of propolis was entirely different from Southeastern and Southern propolis. Neither flavonoid aglycones nor p-coumaric acid were detected in EEP BA. All EEPs demonstrated biological activities against mutans streptococci; EEP BA showed the highest potency in all in vitro parameters evaluated in this study. The ranges of MIC values were 50 (EEP BA)–400 μg/ml (MG), for S. mutans; and 25 (BA)–400 μg/ml (MG), for S. sobrinus and S. cricetus. The bactericidal concentration of EEPs was four to eight times the MIC values. The adherence of S. mutans and S. sobrinus cells and WIG synthesis were markedly inhibited by EEPs, demonstrating significant inhibition at all concentrations compared with the control (80% ethanol) (p < 0.05). EEP BA showed 80% inhibition of cell adherence and WIG synthesis at concentrations as low as 12.5 and 7.8 μg/ml, respectively. The results show that the new variety of propolis was exceptionally effective in all in vitro parameters tested against mutans streptococci; biological effects of propolis are likely not to be due solely to flavonoids and (hydroxy)cinnamic acid derivatives. Received: 14 February 2000 / Accepted: 8 May 2000     

Potent inhibitors of glucagon-stimulated adenylate cyclase associated with serum lipoprotein particles

Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone-H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density lipoprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production

Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1–F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L⁻¹, fraction F1 showed 71% Sf-9 cell growth improvement and 22% β-galactosidase production enhancement over YUF (at 1 g L⁻¹ in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L⁻¹, the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L⁻¹, four other fractions (F2–F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L⁻¹). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.     

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non‐chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30–40‐fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re‐dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans‐membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by‐product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra‐/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642–653, 2017     

Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (<1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml⁻¹ that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.     

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk

Transgenic sheep milk containing the protein human₁-Antitrypsin (AAT) was partitioned in Poly(ethyleneglycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phases at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for -Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.This revised version was published online in October 2005 with corrections to the Cover Date.     

Longitudinal patterns of dissolved organic carbon concentration and suspended bacterial density along a blackwater river

Dissolved organic carbon (DOC) is the dominant form of carbon in transport in blackwater rivers, and bacteria are the major biological agents of its utilization. This study describes longitudinal patterns in DOC concentration and relates them to suspended bacterial populations in the channel. Concentrations of total DOC, three molecular weight fractions, and bacterial numbers were determined at 12 sites along the Ogeechee River in 1985–1986 and 1989 during periods of low and high discharge. Suspended bacterial populations were compared with DOC concentrations to determine if differences in bacterial abundance were related to longitudinal patterns of DOC concentration. Three distinct longitudinal patterns were observed: (1) The longitudinal pattern followed by both total and intermediate molecular weight DOC concentrations was a linear function of the geographic distance along the river. (2) During low flow conditions, there was a high degree of correspondence between patterns of bacterial numbers and low MW DOC (< 1000 apparent MW). (3) During periods of high discharge, the proportion of high (> 10,000) and intermediate (1000–10,000) MW fractions increased, and there was no longer a clear relationship between bacterial cells and low MW DOC.     

Conditions for ethanol precipitation of active 50 S ribosomal subunits from Escherichia coli

Escherichia coli 50 S ribosomal subunits lose activity when precipitated by ethanol from fractions obtained in sucrose density gradient sedimentation. This loss of activity is due to a partial extraction of protein $\text{L7}\text{L12}$ caused by the presence of high sucrose concentrations. Partial extraction also occurs in the absence of added salt or sucrose at low magnesium concentrations. Fully active 50 S subunits can be obtained if the sucrose is diluted to a concentration of 10% or less and the magnesium concentration raised to 20 mm before the addition of ethanol.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The &#103 -glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond &#103 -glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond &#103 -glucosidase. The substrate specificity of the almond &#103 -glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond &#103 -glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

Study on distribution of mycotoxins in cocoa beans

Mycotoxins are not homogeneously distributed in foods which come in naturally small units, such as pistachios and peanuts, and may instead be extremely inhomogeneously distributed due to the occurrence of so-called hot spots. Tests conducted on pistachios, for example, show that a mouldy kernel can be so strongly contaminated with mycotoxins that it has a significant impact on the contamination profile of several thousand kernels. This makes a representative sampling of such foodstuffs very important but also a very difficult task. Whether cocoa beans also have a tendency to form so-called mycotoxin hot spots is hitherto unknown. A miniaturised analysis method was used in tests made on several independent batches of cocoa beans and although these tests showed that the mycotoxins ochratoxin A and the aflatoxins are not homogeneously distributed in cocoa, the tested batches revealed no real hot spots. Presented at the 27^th Mykotoxin-Workshop, Dortmund. Germany, June 13–15, 2005