Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

Identification and quantitation of free and conjugated steroids in milk from lactating women

A method for analysis of metabolic profiles of free and conjugated steroids in milk has been developed. Milk is diluted with aqueous triethylamine sulphate and liquid-solid extraction is achieved on a Sep-Pak C18 cartridge at 60-64 degrees C. Steroids are purified by chromatography on small columns of Lipidex 5000 and sulphohydroxypropyl Sephadex LH-20 [H+] prior to separation into neutral and phenolic compounds, glucuronide, mono- and disulphate conjugate groups on the lipophilic strong anion exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Conjugated steroids are released by enzymatic or solvolytic procedures and separated into a neutral and a phenolic fraction on TEAP-LH-20. The O-methyloxime and trimethylsilyl ether derivatives of the steroids are analyzed by capillary column gas chromatography-mass spectrometry. Fifty steroids were identified in milk collected from women a few days after delivery. Quantitatively about 80% were present as sulphates, 15% as glucuronides and only 5% were unconjugated steroids. The steroid pattern was similar to that in late pregnancy plasma with pregnanolone, pregnanediol and pregnanetriol isomers and dehydroepiandrosterone being predominant. About 10% of the steroid content consisted of estrogens. The total concentration of steroids 2 days after delivery was 20-116 ng/ml, i.e. about 1-5% of the concentration was about 10 ng/ml 1 month after delivery. In one milk sample, collected 2 days after delivery, the steroid concentration (3.6 micrograms/ml) was similar to that in plasma.     

Physiological and structural responses of the cyanobacterium Anabaena cylindrica to aluminium

When adding aluminium (3.7–370 μ M ) as AlCl₃–6H₂0 to cultures of the nitrogen-fixing cyanobacterium Anabaena cylindrica , strain 1403/2a (CCAP), the following responses were observed: The effects of aluminium were dependent on pH. being most drastic at pH 6.0. At this pH the growth of A. cylindrica was significantly reduced by 3.7 μ M aluminium and completely inhibited by 370 μ M . The content of chlorophyll a and phycocyanin decreased after treatment with aluminium. Also, aluminium lowered the rates of both CO₂-fixation and N₂-fixation with total inhibition of both processes by 370 μ M . At the lower concentrations used the nitrogenase activity started to recover after about 100 h. The aluminium content in the cells increased with increasing concentration and with time. At 190 μ M the aluminium concentration in the cells represented 2.4 and 3.3% of the dry weight after 6 and 24 h, respectively. Clogging of filaments and lysis of vegetative cells were apparent at higher aluminium concentrations while the frequency of heterocysts increased in all concentrations used. The most pronounced ultrastructural changes included accumulation of cyanophycin granules and degradation of the thylakoids. The ultrastructure of the heterocysts was however not affected. It is concluded that major reasons for the toxicity are interactions with membranes and phosphate deficiency.     

The response of macroinvertebrates to experimental episodes of low pH with different forms of aluminium,during a natural spate

24 h experimental episodes were created in a soft-water stream in upland Wales, by the simultaneous addition at separate points of sulphuric acid, aluminium sulphate and citric acid. In an upstream reference zone (A) the pH remained above 7, while in the treatment zones, B, C and D successively downstream, it was reduced to c. pH 4.9. Concentrations of filterable aluminium were 0.05 mg l^-1 in Zone B, 0.27 mg l^-1 (0.11 mg l^-1 ‘labile’) in Zone C, and 0.23 mg l^-1 (0.00 mg l^-1 ‘labile’) in Zone D. Chemical manipulation coincided with a spate, during which flow increased from 0.02 m³s ^-1 to a maximum of 0.07 m³s^-1. Only the ephemeropteran B. rhodani showed a drift response: drift density was not affected by flow (Zone A) or by organically bound aluminium (Zone D), but increased approximately 6-fold in both the acid (B) and ‘labile’ aluminium (C) zones; its benthic density declined significantly in Zones C and D, by 78% and 89% respectively. We relate these results to the biological importance of aluminium chemistry during natural acidic episodes.     

Aluminium toxicity during regular haemodialysis.

In the west of Scotland the incidence of dialysis encephalopathy has been confined to three geographical areas where the concentration of aluminium in the water supply is greatly increased owing to the addition of aluminium sulphate. Eight patients with encephalopathy who dialysed at home in these areas had greatly increased serum aluminium concentrations, and a significant correlation was found between serum aluminium concentrations and the concentrations of aluminium in the water supply. This study provides further evidence that the dialysis encephalopathy syndrome is due to aluminium intoxication, the major source of aluminium being the water supply from which dialysis fluid prepared.     

Hybridoma cultures using porcine serum not containing immunoglobulin and dialysis fractions

The whole swine serum was treated with ammonium sulphate to precipitate immunoglobulins. The remained IgG was removed with the use of protein A-sepharose. The hybridoma cells producing monoclonal antibodies to lambda phage (class IgG) were cultured in Dalbecco's modified Eagle medium with addition of a 5% whole swine serum or of a treated unwhole one (final concentration of the protein being 3 mg/ml). Upon these conditions, hybridoma cells had similar growth rate and population density (1-1.3 X 10(6) cells/ml). Maximal antibody concentration was almost similar (80-90 mcg/ml). Purity of a sample of monoclonal antibodies isolated by the method of chromatography with the use of protein A-sepharose from supernatant containing the unwhole serum was no less than 99%, whereas it was considerably lower (12-15%) in the case of the whole serum.     

Effects of sulphate and pH on the plant-availability of phosphate adsorbed on goethite

The adsorption of phosphate on metal (hydr)oxides may be influenced by the pH and by the adsorption of other ions. In this study, the influence of sulphate and pH on phosphate adsorption on goethite and the availability to plants of adsorbed phosphate was examined. Maize plants were grown on suspensions of goethite with adsorbed phosphate, containing the same total amount of phosphate and either 0.11 mM or 2.01 mM sulphate at pH 3.7, 4.6 or 5.5. The uptake of phosphorus by the plants increased with the larger sulphate concentration and decreasing pH. Mean P uptake in the treatment with 2.01 mM sulphate and pH 3.7 was 55 µmol plant^-1, whereas in the treatment with 0.11 mM sulphate and pH 5.5 it was 2 µmol plant^-1. Batch adsorption experiments using³² P and speciation modelling of ion adsorption showed that in the presence of sulphate, the phosphate concentration in solution strongly increased with decreasing pH, due to competitive adsorption between sulphate and phosphate on goethite. Modelled phosphate concentrations in solution in the uptake experiment were all below 0.6 µM and correlated well with the observed P uptake. This correlation indicates that the strong influence of the sulphate concentration and pH on the plant-availability of adsorbed phosphate results from the competition between sulphate and phosphate for adsorption on goethite.     

Glycosulphatase from Pseudomonas carrageenovora. Purification and some properties

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55 000. Conditions of optimal sodium chloride concentration and pH at 25 degrees C were 0.25--0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-D-galactose 4-O-sulphate].     

Aluminium speciation in natural water by sorption on a complexing resin

Very stable aluminium complexes may be present in natural waters, which can be detected only using appropriate methods. One of them is the resin titration based on the sorption of aluminium on a strongly sorbing resin, Chelex 100. It was here used to detect strong aluminium complexes, and to characterize them by determining their concentration, and the corresponding stability constant. High and low salinity waters were sampled in different sites in the North of Italy. In all the samples aluminium complexes with high stability constant, up to 10(17.4) M(-1) in the less acidic solution, were detected. The stability constant depends mainly on the solution acidity, increasing with increasing pH up to 7. The concentration of the ligands responsible for the strong complexation is similar to that of aluminium (from 0.5 to 1.5 microM), or somewhat lower in the case of estuarine and sea waters. A small fraction of aluminium (from 0% to 2%) in freshwaters, higher in estuarine and sea waters (14% and 10%, respectively), is present in weakly bound forms which could also be the hydrolysis products. The conditional constants of the strong complexes were determined for the different samples examined. They were found to be slightly lower in the case of the high salinity waters, in which a value of 10(16.1) M(-1) at pH 7.5 was obtained. This is probably due to the higher ionic strength in marine water, which strongly influences the complexation of trivalent metal ions, as seen for example also in the hydrolysis. It could be deduced that similar substances, but at different concentration, would be responsible for the aluminium complexation in the sea and freshwaters here examined. They could be natural organics like fulvic substances, or better some particular complexing sites in this substances with very high affinity for aluminium.     

Heparan sulphate-degrading endoglycosidase in liver plasma membranes.

An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.     

Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp

Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40-60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40-100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5-11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 degrees C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 degrees C after 30 min.     

Virus detection in soils: a comparison of four recovery methods.

Among nine eluents tested, 0.5% (w/v) isoelectric casein at pH 9.0 and 0.5% (w/v) non-fat dry milk (pH 9.0) were the most efficient in eluting poliovirus type 1 (Sabin) from Eustis fine sand. However, no significant difference was found between the overall (elution followed by concentration) virus recoveries by non-fat dry milk, isoelectric casein, beef extract, and glycine-EDTA methods. High overall recovery (75%) of low input (200 PFU) of viruses from 100 g of soil was achieved by the isoelectric casein method. It was found that the recovery efficiency of this method was not significantly affected by the soil type, following examination of four Florida soils. The mean overall recovery for the four soils was 50%. For other enteroviruses, the overall recovery for coxsackie B3 was 88% but was significantly lower (23%) for echovirus 4. Examination of the efficiency of the casein method under field conditions showed that it was possible to recovery low poliovirus numbers from soil (0.9-1.3 PFU/g soil).     

Chalcopyrite concentrate leaching with biologically produced ferric sulphate

Biological ferric iron production was combined with ferric sulphate leaching of chalcopyrite concentrate and the effects of pH, Fe3+, temperature and solids concentration on the leaching were studied. The copper leaching rates were similar at pH of 1.0-1.8 and in the presence of 7-90 g L-1 Fe3+ despite massive iron precipitation with 90 g L-1 Fe3+. Increase of the leaching temperature from 50 degrees C to 86 degrees C and solids concentration from 1% to 10% increased the copper leaching rate. Increase in solids concentration from 1% to 10% decreased the copper yields from 80% to 40%. Stepwise addition of ferric iron did not improve the copper yields. CuFeS2, Ag and Cu1.96S potentials indicated the formation of a passivating layer, which consisted of jarosite and sulphur precipitates and which was responsible for the decreased leaching rates.     

Treatment and clarification of fermented broth in bacterial enzyme production

Summary Acetic acid, calcium chloride, aluminium sulphate and polyacrylamide were tried for flocculation and settling of suspended solids. Adjustment of pH, centrifugation, precoat filtration with filter aid were studied for clarification of fermentation broth in alpha-amylase production. Combination of aluminium sulphate and polyacrylamide as flocculating agents and precoat filtration was found to be the best strategy for alpha-amylase enzyme recovery from the broth.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases.

Assay methods were developed enabling separate determination of N- and O-sulphotransferase activities in an enzyme preparation from mouse mastocytoma. N-Desulphoheparin and chemically N-acetylated heparan sulphate were used as specific exogenous sulphate acceptors in the transfer of [35S]sulphate residues from adenosine 3'-phosphate 5'-[35S]sulphatophosphate to amino and hydroxyl groups respectively. The resulting 35S-labelled polysaccharides were isolated as their cetylpyridinium complexes on filter paper. Sulphotransferases were solubilized from a mastocytoma microsomal fraction by treatment with detergent-alkali. The pH optimum for both enzymes was about 7.5 Km with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 X 10(-5) M for the N-sulphotransferase and 1 X 10(-4) M for the O-sulphotransferase(s). The enzymes required bivalent cations for maximum activity, Mn2+ stimulating both the N- and O-sulphotransferase four- to five-fold, whereas Ca2+ increased the N- but not the O-sulphotransferase activity. The O-sulphotransferase was found to be more sensitive to heat-inactivation, 60% of the activity being lost after 1 min at 50 degrees C, whereas only 15% of the N-sulphotransferase activity was lost. In contrast, the N-sulphotransferase was selectively inhibited (or inactivated) by NaCl; at 0.125 M-NaCl concentration the O-sulphotransferase activity was essentially unaffected, whereas the N-sulphotransferase activity was depressed by 80%. These results strongly indicate that N- and O-sulphate-transfer reactions should be ascribed to different enzymes, or, alternatively, to separate and independent active sites on the same enzyme molecule.     

The inositol 1,4,5-trisphosphate receptor binding sites of platelet membranes. pH-dependency, inhibition by polymeric sulphates, and the possible presence of arginine at the binding site.

The present study was initiated to characterize the inositol 1,4,5-trisphosphate (InsP3)-binding site in human platelets that is involved in Ca2+ release. InsP3 binding to platelet membranes was measured in two ways; (1) by displacement of labelled InsP3 with unlabelled InsP3, as in previous studies, and (2) directly, using only radioactive InsP3 as ligand, over the concentration range 0.25-100 nM. At physiological pH (7.1) the binding data were best fitted by a model for a single saturable binding site, with KD = 11.8 nM and Bmax. = 1.4 pmol/mg of protein. At alkaline pH values (8.3 and 9.4) binding was best fitted by a two-site model, the second site being of higher affinity (KD = 0.75-1.2 nM) but lower concentration (Bmax. = 0.195-0.6 pmol/mg of protein). All binding of InsP3 was blocked by polymeric sulphates (heparin, dextran sulphate, polyvinyl sulphate) regardless of pH. The specific arginine-modifying reagent p-hydroxyphenylglyoxal irreversibly blocked InsP3 binding, suggesting the presence of arginine at the recognition site for InsP3 binding. NN'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (ECCD), which are carboxy-group-specific reagents, blocked Ca2+ release, but not InsP3 binding, indicating the existence of another site that regulates Ca2+ release apart from the active centre for InsP3.     

Isolation of bacteriocins through expanded bed adsorption using a hydrophobic interaction medium

Two lactic acid bacterium bacteriocins were isolated from fermentation medium through expanded bed adsorption using a hydrophobic interaction gel. First, amylovorin L471, produced by Lactobacillus amylovorus DCE 471, was selected for the optimisation of the loading and eluting conditions. Secondly, the results of the optimisation were applied for the isolation of enterocin RZS C5, a bacteriocin produced by Enterococcus faecium RZS C5. Optimal adsorption was obtained for a medium with concentration of 1.0 M ammonium sulphate and adjusted to pH 4.0 (94.9% for amylovorin L471 and 75.0% for enterocin RZS C5). Elution with 50% ethanol, buffered at pH 6.0, resulted in an optimal total recovery of the bacteriocin activity of 47.6 and 57.6%, respectively. The highest fold purification expressed as the increase in specific activity (AU/mg) corresponded to the highest recovery, being 140- and 1677-fold, respectively. Nevertheless, a total recovery of only 25.6% with an increase of the specific activity of 121 times was obtained after conventional isolation by ammonium sulphate precipitation.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.