Buffered Saline Extract Research Articles

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A galactose-specific lectin was purified from the skin mucus of African catfish (Clarias gariepinus) and the physicochemical properties determined. Phosphate buffered saline extract of the skin mucus of African catfish specifically agglutinated erythrocytes of rabbit and human blood group B, but did not agglutinate bat, rat, hen and human blood A and O erythrocytes. The haemagglutinating activity of the lectin was completely inhibited by lactose while galactose and melibiose inhibited the activity to some extent and was calcium-independent. The purified lectin has a native and subunit molecular weight of 63, 000 and 20, 000 Daltons, respectively suggesting a homotrimeric structure for the protein. The protein contained 126 amino acid residues per subunit. This was characterized by large amount of polar amino acids that constituted about 60% of the total amino acids. The lectin showed maximum activity over the pH range 6 – 9 and was heat stable up to 50°C. Ethylenediaminetetraacetic acid (EDTA) had no inhibitory effect on its haemagglutinating activity. Periodic acid–Schiff (PAS) staining showed that the lectin was not a glycoprotein. Chemical modifications of serine and arginine residues of the protein did not affect its haemagglutination activity while modifications of cysteine, tryptophan and histidine residues led to total loss of its activity. The study concluded that African catfish skin mucus lectin exhibited similar physicochemical properties with lectins from other fish skin mucus. Key words: African catfish, Lectin, haemagglutination, skin mucus, galactose-specific, Clarias gariepinus

Extraction Of House Dust Mite Allergen, Der P1, From Dust

A recent article in this journal found that the type of extraction buffer did not affect measured levels of the major group one allergen (Der p 1) from the house dust mite, Dermatophagoides pteronyssinus, in household dust samples (1). This differed from a previous study where about twice the levels of Der p 1 were found in borate-buffered saline extracts, compared to ammonium bicarbonate buffer or phosphate-buffered saline extracts, irrespective of extraction temperature or time of extraction (2). In discussing our previous study (2), the authors state that we did not use Tween-20 in our extraction buffers. Although our article did not make a mention of Tween-20, we in fact did use it in our extraction media. It was an oversight of us in that we did not mention this in our article. We further explored temperature and buffer effects on Der p 1 extraction in an external quality control program where six dust samples were sent to 23 laboratories worldwide (3). Results from this study again showed that type of extraction buffer and extraction temperature influenced Der p 1 measurements. The majority of the participant laboratories did use Tween-20 as a wetting agent in their extraction buffers. As pointed out by the authors, adding Tween-20 to extraction media results in higher endotoxin levels and is recommended by the European Committee for Standardisation. The authors call for further investigation on the effects of Tween-20 on extraction effi ciency of all indoor allergens from dust. However, the majority of indoor allergen analysis laboratories use ELISA or MARIATM kits from Indoor Biotechnologies where the recommended extraction medium is phosphate-buffered saline containing 0.02 % Tween-20. It would take much effort to study this systematically to obtain results with suffi cient internal and external validity.

The pruritogenic effect of Anaphe venata extracts in rats: the role of cholinergic, GABAergic and opioid systems

This study sought to investigate the effect of the crude aqueous and phosphate buffer saline (PBS) extracts of Anaphe venata on body scratching behaviour in rat in a novel environment and also to determine the neural mechanism(s) involved. Aqueous and PBS extracts of Anaphe venata were prepared and their effects on body scratching behaviour were evaluated in rats. Animals were divided into four groups (n = 6-12 per group) and graded doses of extracts (100-400 mg/kg) were administered (dissolved in normal saline) intraperitoneally (i.p.) to each animal in the experimental groups. The control group received an equivalent volume of normal saline. Behavioural scores were recorded for a period of 30 minutes after the administration of normal saline or extract. The role of various receptors in the extract induced pruritus was evaluated using known receptor agonist/antagonists. Results showed that aqueous Anaphe extract induced dose-dependent increase in body scratching behaviour (p

Antimicrobial studies of Cosmos caudatus Kunth. (Compositae)

The present study aims to investigate crude n-hexane, diethyl ether (Et2O), ethanol (EtOH) and phosphate buffered saline (PBS) extracts of Cosmos caudatus Kunth leaves for their antimicrobial potential against 5 microbial strains comprising 2 Gram positive bacteria: Bacillus subtilis, Staphylococcus aureus, 2 Gram negative bacteria: Escherichia coli, Pseudomonas aeruginosa and 1 fungi: Candida albicans by the disc diffusion method. Preliminary antimicrobial screening showed inhibition by the n-hexane, diethyl ether, and ethanol extracts against all the tested microbes, however the phosphate buffered saline extract was inactive against Bacillus subtilis and Staphylococcus aureus. MIC values ranging from 6.25 - 25 mg/ml for the tested crude extracts were obtained by the microtiter plate method. This study showed that the crude extracts of the leaves of C. caudatus Kunth. could be potential source of new antimicrobial agents especially to treat infections caused by the tested microbial strains and confirms its utility in folk medicines. Key words: Cosmos caudatus Kunth., antimicrobial activity, gram positive, gram negative bacteria, fungi, MIC.

Photoluminescent Distinction among Plant Life Forms Using Phosphate Buffered Saline Extract Solutions

Photoluminescence of plant extract solutions has been investigated for discrimination of plant life forms (grasses, forbs, and shrubs) using principal component analysis (PCA). Clippings from each of six plant species representing three different plant life forms potentially found in the diets of free-ranging herbivores in the Chihuahuan Desert of North America were investigated for possible discernment. These plants included Sporobolus flexuosus (mesa dropseed, a grass), Pleuraphis mutica (tobosa, a grass), Dimorphocarpa wislizenii (spectacle pod, a forb), Sphaeralcea incana (pale globemallow, a forb), Flourensia cernua leaves (tarbush, a shrub), and Atriplex canescens leaves and stems (fourwing saltbush, a shrub). Emission spectra (370-600 nm) from phosphate buffered saline (PBS) extract solutions (pH 2.2, 7.5 and 12.5) were recorded for each plant with excitation at 365 nm. Use of PBS minimized chlorophyll interference. Discernment among plant life forms within these plant species was achieved.

Vitamin B1 and B2, dietary fiber and minerals content of Cruciferae sprouts

The contents in selected Cruciferae seeds and ready-to-eat sprouts of thiamine (B1) and riboflavin (B2) were determined by HPLC methodology. The content of soluble and insoluble fractions of dietary fiber was determined by the enzymatic method. In addition, the calcium, magnesium, zinc, cooper, ferrum and manganese concentrations were determined by atomic absorption spectrometry and after that the correlation between some mineral content and the ability of seeds and sprouts phosphate buffered saline extracts to scavenge the superoxide anion radicals in vitro was investigated. The small radish, radish, rapeseeds and white mustard seeds contained vitamin B1 in the range from 0.41 up to 0.70 mg/100 g d.m., however its amount found in the ready-to-eat sprouts were lower by 46, 39, 42 and 47%, respectively. In contrast, the content of vitamin B2 in the ready-to-eat sprouts showed approximately three-fold higher content when compared to its range found in the seeds (0.096 mg/100 g d.m up to 0.138 mg/100 g d.m.). The total dietary fiber content in ready-to-eat sprouts, including the soluble and insoluble forms, was 20% higher when compared to the seeds and the proportion of insoluble to soluble fiber was about two-fold higher in radish sprouts, four-fold higher in rapeseed sprouts, and six and nine-fold higher in small radish and white mustard sprouts, respectively. The sprouts contained higher amounts of Ca, Mg, Cu and Zn approximately by 12, 14, 25 and 45%, respectively, when compared to the seeds. The similar beneficial changes were noted for Cu and Zn. Their amount noted in sprouts was higher by average of 25% for Cu and by 45% for Zn. No changes in Mn and Fe levels were found between seeds and sprouts. One exception was only made to Fe content in the white mustard sprouts in which the Fe amount was lower than that found in the seeds. The SOD-like activities of the seed extracts were positively correlated only with the manganese level (r=0.94), however, this correlation was not found in ready-to-eat sprouts. No other correlations were found between SOD-like activity and microelements contents in the seeds and sprouts.

Essential Components of Antimicrobial Gastrointestinal Epithelial Barrier: Specific Interaction of Mucin with an Integral Apical Membrane Protein of Gastric Mucosa

The gastric mucosal protective barrier consists of two essential elements: mucus glycoprotein, mucin, secreted by gastric mucosal cells, and the mucin binding protein (MBP), an integral component of the apical epithelial membrane. The studies described here provide evidence on the structure of MBP, its interaction with mucin, and the susceptibility to phospholipase C (PLC) and Helicobacter pylori protease. The rat gastric mucosa was used to isolate mucin and the apical epithelial membranes. A buffered saline extract of the mucosal cells was used for the isolation of mucin and the 1% Triton X-100-insoluble gastric apical membranes for the preparation of MBP. The studies on MBP, the mucosal mucin receptor revealed that the protein is anchored in apical membrane through glycosylphosphatidylinositol (GPI). The deamination of MBP with nitrous acid afforded phosphatidylinositols (PIs) and a water soluble, 97 kDa glycosylated protein. The in situ studies with untreated rat gastric mucosa and the mucosa depleted of mucin showed that MBP without mucin was susceptible to the proteolytic degradation with pepsin and H. pylori proteases, but was not released from the apical membrane by the treatment with bacterial PLC. The study of carbohydrate ligands for MBP revealed binding of octa- and decasaccharides of gastric mucin. The severe impairment in mucin adhesion to MBP, induced by the diet containing ethanol, supports the conclusion that specific carbohydrate determinants participate in mucin attachment to MBP and epigenetic control of the processes that coordinates its interaction with apical mucosal epithelium in the formation of innate protective barrier.

Physico-chemical characterization of grain dust in storage air of Bangalore.

An Anderson personal cascade impactor was used to study the particle mass size distribution in the storage air of two major grain storage centers in Bangalore. Dust levels in storage air as well as the personal exposures of workers were determined along with a detailed study on the particle size distribution. Protein and carbohydrate content of the dust were also determined respectively in the phosphate buffer saline (PBS) and water extracts by using the standard analytical techniques. Personal exposures in both of the grain storage centers have been found to be much above the limit prescribed by ACGIH (1995-96). But the results of particle size analysis showed a higher particle mass distribution in the non-respirable size range. The mass median diameters (MMD) of the storage air particulate of both the centers were found to be beyond the respirable range. Presence of protein and carbohydrate in the storage air dust is indicative of the existence of glyco-proteins, mostly of membrane origin.

Studies on Bioactivity of the Root Extract of Plumbago zeylanica

The phosphate buffered saline extract of the roots of Plumbago zeylanica was investigated for anti-inflammatory activity. The extract stabilized red blood cells subjected to both heat and hypotonic induced lyses. The extract exhibited a biphasic response. The enzymatic activities of both alkaline and acid phosphatases were reduced, while adenosine triphosphatase activity was stimulated in the liver homogenates of formaldehyde induced arthrititic rats. A possible anti-inflammatory action of the extract of P. zeylanica is, therefore, speculated.

Efficacy of various vaccines against pneumonic pasteurellosis in cattle: a meta-analysis

The objective of this study was to compare the protective efficacy induced by various experimental and commercial vaccines used in the past 12 years (1982–1994) against bovine pneumonic pasteurellosis. All studies using an experimental-challenge model to evaluate protective efficacy induced by vaccine preparations were identified using a combination of VETCD, CAB, and MEDLINE searches. A total of 72 trials were identified in 27 studies. Age groups of animals used, sample sizes used, vaccine types, route of vaccination, dose of vaccine, adjuvant types, booster times, time of challenge since first vaccination, challenge methods, challenge doses, challenge route, observation times postchallenge, and raw mean lung-lesion scores (defined as percent lung involved) were extracted from each publication and used in the analysis. Vaccine types were grouped into 11 categories as controls, live, potassium thiocyanate (KSCN) extract, phosphate buffered saline extract (PBSE), 30 kDa recombinant protein, bacterin, streptomycin dependent live (Sm D live), sodium salicylate extract (SSE), sarkosyl outer membrane protein extract (sarkosyl OMP), or culture supernatant (CS) categories based on the subunit content in each preparation. In a second analysis, the chemically altered live vaccine was grouped together with live Sm D group as modified live vaccines. The leastsquares means of the postchallenge lung-lesion scores of each vaccine type were then compared after controlling for all the above-mentioned variables in an analysis of variance using Fischer's protected least significant difference. The statistical analysis was done using the general linear models procedure of Statistical Analysis Systems Institute Inc.. Protective indices for each vaccine type were calculated as a function of independent probabilities of protection among vaccinates and controls. The protective indices of each vaccine type were then compared in a similar model. Cattle vaccinated with live vaccines had the smallest lung-lesion scores (9 ± 8) which significantly differed from that of controls (44 ± 8), bacterins (23 ± 6), and live Sm D (38 ± 12) or modified live (31 ± 10). KSCN extract (9 ± 10), bacterin, and CS (20 ± 9) also had significantly lower ( P < 0.05) lung-lesion scores compared to controls. The CS and KSCN vaccines showed a significantly ( P < 0.05) better protective index (PI) (0.70 and 0.68 respectively) than bacterins (0.50) or bacterin + CS (0.44) vaccines. Saline extract and live vaccines had better ( P < 0.05) PI (0.60 and 0.70 respectively) compared to bacterins.

Specific and Cross-Reacting Antibodies in Human Responses to Onchocerca volvulus and Dracunculus medinensis Infections

Immunoelectroblotting and enzyme-linked immunosorbent assay were used to identify non-cross-reacting antigenic components of Dracunculus medinensis and the filarial worms Onchocerca volvulus, Loa loa, Wuchereria bancrofti, Brugia malayi, and Mansonella ozzardi. Parasite specific serodiagnostic ELISA systems for onchocerciasis and dracunculiasis were devised based on these findings. Phosphate buffered saline extracts of adult worms were passed through a column of monoclonal antibodies to phosphorylcholine (PC). Crude and PC-depleted extracts were reacted on ELISA plates with individual sera from subjects infected with a range of nematodes. Binding of total antibody (Ig) or IgG class antibody and IgG4 subclass antibody was revealed using goat antihuman-Ig-phosphatase conjugate, or appropriate mouse monoclonal antihuman-Ig-type-specific reagents, followed by goat antimouse-Ig-phosphatase conjugate. Specificity of ELISA was improved by restricting reaction to the host's IgG4 antibody subclass, and/or by removing PC determinants from crude antigens. In parallel immunoelectroblots, crude and PC-depleted extracts probed with pooled sera showed potentially useful diagnostic antigens, including a 12 kDa protein from D. medinensis and 14, 18, and 27 kDa proteins from O. volvulus. Two Onchocerca specific ELISA systems non-reactive with antibodies to D. medinensis were devised.

Influence of immune treatments against Dirofilaria immitis infection in dogs.

Mongrel dogs were inoculated with two kinds of antigenic substances. The first was a phosphate buffered saline extract of whole Dirofilaria immitis mixed with aluminum hydroxide gel (group 1), and the second was an orally administered live Metastrongylus apri infective larvae (L3) (group 2). Both groups were then infected with D. immitis L3. Indirect hemagglutination (IHA) tests showed that the antibody was produced by these inoculations before the infection was introduced, even in dogs inoculated with M. apri. This suggests a cross-reactivity between D. immitis and M. apri. The initial passive cutaneous anaphylaxis (PCA) antibody production was markedly delayed by about 70 days in group 2 compared with the production in the infected control dogs (group 3). The appearance of microfilaremia was also delayed by about one month in group 2 compared with that in the above control group. All dogs were sacrificed after the termination of the observation and worms recovered from the right ventricle and pulmonary arteries were counted and measured. The results indicated that immunization resulting from the homologous worm-somatic antigen might accelerate the growth of the infected larvae, whereas immunization resulting from the heterologous worm antigen, but cross-reactive to D. immitis, might disadvantageously affect the growth.

Haemagglutinating and Trypsin Inhibitor Activities of Lupin Seed (Lupinus angustifolius)

ABSTRACTThe phosphate buffer saline (PBS) extracts of defatted lupin seed flour showed haemagglutin activity only with trypsinized rabbit erythrocytes. The lectin tested in crude extracts showed specificity towards galactose and lactose. Heat stability studies of PBS extracts on incubation for 10 min showed no reduction of haemagglutinin activity in the temperature range 40°C to 80°C while no haemagglutination was observed when incubated at 90°C for 8 min. The trypsin inhibitor activity of flour extracts was 14.4 TUI/mg protein.

Purification and physico-chemical properties of lectins from the jack fruit (Artocarpus iutegrifolia)

A haemagglutinating material was isolated and purified from the phosphate buffered saline extract of the seeds of the jack fruit using an immobilized N-aeetyl-D-galactosamine column. This material was composed of two iso-lectins of molecular masses 11 500 and 15 000. The lectins agglutinated native washed red blood cells of the human A, B and 0 groups and sheep, rabbit and mouse erythrocytes. The lectins were found to be composed of single polypeptide chains and they contained no covalently linked sugars. The lower molecular mass material was present in considerably greater quantity than the higher molecular mass component. On isoelectric focussing on PAG the lectins gave a spread of components with calculated pI between 6.0 and 8.3.

Schistosoma mansoni: Vaccination with adult worm antigens

Under relatively mild conditions it has been possible to release from 5. mansoni, large amounts of antigens. Immunization experiments performed in rabbits with this phosphate buffered saline extract of adult worms (Saline Extract, SE) in either Complete Freunds Adjuvant or Corynebacterium parvum have resulted in very high levels of protection (76–99%) upon challenge infection of the immunized rabbits. Furthermore, fractionation of SE by gel chromatography, permitted the isolation of a purified fraction (FI) that also induced protection against challenge infection. FI appeared to contain most of the protective antigens of SE. The present data report the evaluation of different parameters related to the immunization, purification and biochemical analysis of SE.

Fractionation of antigen for ELISA of bovine fascioliasis

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis; Mean+2 x SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+/-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

Lectins and the soybean- Rhizobium symbiosis : I. Immunological investigations of soybean lines, the seeds of which have been reported to lack the 120 000 dalton soybean lectin

Seeds of six soybean lines ( Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunofiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lactin could be purified by affinity chromatography on Sepharose- N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography. Phosphate buffered saline extracts of roots, hypocotyls, stems, and leaves of 3–66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 μg soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were modulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.

Sources of larval salivary gland secretion in the dipteran Chironomus tentans.

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.

Complement fixation reaction and agar gel double diffusion test in Tyzzer's disease of mice.

Complement fixation reaction and agar gel double diffusion test were made using as antigens, water or phosphate buffer saline extracts of liver from mice severely infected with Tyzzer's disease. The supernatant of the extract heated at 56 C for 30 minutes was highly reactive with immune mouse serum in the complement fixation test, while an intense precipitate band was observed only with unhealed extracts in agar gel double diffusion tests. Cross reactions were observed in both tests between liver extracts from mice infected with different strains of organisms originating from different sources and showing varied degrees of virulence. Such reactions were not shown between Tyzzerimmune serum and liver extracts from uninfected or murine hepatitis-infected mice, nor were they shown between the Tyzzer-antigen and normal or anti-murine hepatitis virus serum. The complement fixation test was used to test retired breeder mouse sera for disease contamination in mouse colonies. Positive reactions were observed in test sera collected from some breeding colonies that subsequently proved to be contaminated by the cortisone provocation test in young animals.

Immunochemical study of the antigens of Trichinella spiralis larvae. II. Some physicochemical properties of these antigens

A study has been made of some physicochemical properties of the precipitating antigens, of Trichinella spiralis larvae using immunoelectrophoresis in agar as the principal method of analysis. Evidence is presented which strongly suggests that the isoelectric point of the major antigen of these parasite larvae is similar to that of normal human serum gamma-globulin. Fractionation of pH 7.0 buffered saline extracts of these parasites with common laboratory reagents (5% trichloroacetic acid, acetone and ammonium sulfate at 50% saturation) has indicated that ten of the eleven antigens of this parasite are proteins. Evidence is also presented which suggests that one of the ten protein antigens, may also contain some polysaccharide associated with the protein molecule. These fractionation studies have been unable to clarify the chemical nature of the major precipitating antigen of these larvae.