Saponin Solution Research Articles

Método fluorescente (diacetato de fluoresceína e brometo de etídeo) para o estudo da viabilidade de Cryptococcus neoformans em líquor

The utilization of the fluorescent method (fluorescein diacetate DF and ethidium bromide BE), to verify the viability of fungal cells, was studied in 40 samples of liquor, from patients with neurocryptococcosis. For removing leukocytes and red blood cells, which produce interfering fluorescence, good results were obtained with 0.3% saponin solution. After processing of liquor, 0.1 ml aliquots of resulting suspension were mixed to equal volumes of fresh DF-BE solution. The best incubation period for staining was 30 minutes, resulting in good differentiation between viable (green fluorescence) and non viable (red fluorescence) cells.

Systemic delivery of insulin through eyes to lower the glucose concentration.

An attempt has been made in this study to develop a simple, economical and painless method for the delivery of insulin systemically without using parenteral routes. Insulin was shown to be absorbed effectively into systemic circulation through the eyes. With 0.125% (0.031 mg/25 microliters), 1% (0.25 mg/25 mg/25 microliters) insulin instilled topically into the eyes, systemic blood concentrations of 1.3 ng/ml, 9 ng/ml, and 24 ng/ml, respectively, could be attained. When 25 microliters of 1%, 2% and 5% insulin plus 1% saponin solution were instilled into eyes they reached 63 ng/ml, 89 ng/ml and 195 ng/ml, respectively in the systemic circulation. These results indicate that the systemic absorption of 1% insulin through the eyes can be enhanced at least 7-fold when 1% of the surfactant, saponin, was added to the solution. However, the insulin absorption was not affected by aminopeptidase inhibition nor by the change of pH in the range of 5 to 8. Most importantly, the blood glucose was reduced concomitantly with the increase of blood insulin when 50 microliters of 1% insulin in 1% saponin solution was instilled in the eyes. The blood glucose of alloxan-treated diabetic animals reduced drastically from 370 mg % to 185 mg % in 90 min and further to 75 mg % in another 150 min. In normal animals, 25 microliters of 1% insulin in 1% saponin reduced blood glucose from 102 mg % to 50 mg % in an hour and blood glucose remained low for at least 3 hrs. These results indicate that insulin can be delivered systemically through the eyes, particularly with the surfactant enhancers, such as saponin. This new method is economical and simple as compared with parenteral administration and/or minipump implantation. It is hoped that this new method would improve patients' compliancy in using insulin for diabetes treatment.

A Perfusion Method Using Low Concentration of Glutaraldehyde and Saponin Solution for the Observation of the Cytoskeleton of Epithelial Cells

A Perfusion Method Using Low Concentration of Glutaraldehyde and Saponin Solution for the Observation of the Cytoskeleton of Epithelial Cells

A new approach to the biochemical pathology of the vascular system, using time governed laminar elution and bioluminescence analyses.

Endothelial cells which cover the inner wall of blood vessels were extracted for bioluminescence analyses of nucleotides and enzymes. The contaminating blood was removed by heparinization and rinsing with ammonium chloride. The content of the endothelial cells of the rat aorta was reached by time governed laminar elution, using a saponin solution to disrupt the cell membranes. Uniformity of extraction was achieved with a Hamilton programmable pump. All the analyses of the eluted fractions showed a characteristic and reproducible peak. The activities of glucose-6-phosphate dehydrogenase and adenylate kinase were significantly higher in the diabetic animals whereas the amount of nucleotides did not differ between diabetic and control rats. The laminar elution technique combined with bioluminescence assay represents a new approach to studies of biochemical alterations in the endothelial cells. The method is also useful for extraction and analyses of other surface layers.

Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.

Dense line in cisterna of rough-surfaced endoplasmic reticulum of hepatocytes of the rat perfused with saponin solution

The effect of brief perfusion of saponin solution to the membrane of rough-surfaced endoplasmic reticulum(r-ER) was observed in hepatocytes at electron microscope level. The liver of rats was perfused with 1% saponin solution in 0.2m phosphate buffer (pH 7.0) for 3 min. then perfused again with 4% glutaraldehyde buffered with 0.1m sodiumcacodylate-HCl at pH 7.4. The most striking alteration of ultrastructure of hepatocytes was the dense intracisternal line of the r-ER. That dense line often had cross striation or “ladder structure”. Although its functional significance remains to be cleared, it should reflect the denature of membrane structure of r-ER as one of effects of saponin.

Isolation of focal contact membrane using saponin

The fragments of lower cell surface remained attached to the substrate after incubation of mouse or chick fibroblasts in 0.2% saponin solution and subsequent removal of cells under the action of shearing force. These fragments corresponded exactly to the cellular focal contacts seen by interference reflection microscopy. Ultrastructurally they were membrane fragments with typical three-layered structure. No cytoskeletal components were found in saponin-isolated focal contact membranes either by immunofluorescence or electron microscopy. Only one major cell-derived protein with an apparent molecular weight (MW) of 51 kD (chick embryo fibroblasts) or 47 kD (mouse embryo fibroblasts) remained on the substrate after saponin treatment and removal of cells.

Enrichment and partial enzyme characterization of ATPase activity associated with the outward-facing membrane complex and inward-facing membrane of the surface epithelial syncytium of Schistosoma mansoni

The outward-facing (OFM) and inward-facing (IFM) membranes of the surface epithelial syncytium of Schistosoma mansoni were separated by sequential exposure to saponin solutions. The OFM, containing both inner and outer bilayers, contained ATPase activity that was stimulated by Mg 2+ and Na +, but not K + or HCO 3 −, and was inhibited by Ca 2+ and ethacrynic acid. The OFM enzyme was unaffected by ouabain, oligomycin, SCN − and azide and had a pH optimum of 7.5. The OFM ATPase therefore has properties similar to ATPases characterized from the apical membrane of a variety of epithelial cells where it is thought to augment the regulatory cell volume decreasing function of (Na ++K +)Mg 2+−ATPase. The IFM contained ATPase activity that was stimulated by Mg 2+, Na + and K +, and was inhibited by ouabain indicating that the IFM enzyme was the Na +-pump ATPase. The results are discussed in terms of the transepithelial transport function of the surface epithelial syncytium and a Ca 2+-ATPase reported previously from the OFM of S. mansoni.

A quick and simple method for the routine determination of acetyl- and butyrylcholinesterase in blood.

The salient features of this method for biological monitoring of occupational exposure to organophosphorus insecticides are: (a) acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are determined separately in whole haemolysed blood using specific substrates at appropriate concentrations; (b) 20 microliter of blood drawn from the finger tip is sufficient for both determinations; (c) the blood sample is immediately diluted with a solution of saponin and may thereafter be frozen for storage; (d) diagnostic kits, commercially available for the determination of plasma BuChE, may be employed with modifications; (e) the kinetic procedure is avoided by blocking the enzyme reactions at the end of the incubation period. This paper describes attempts to achieve optimal conditions for the two reactions. Under the conditions finally chosen, the whole blood 'AChE' activity value still includes a small percentage of plasma BuChE activity (12.5% of the total), while the whole blood 'BuChE' activity includes a small percentage of erythrocyte AChE activity (7% of the total). Results of determinations performed with this procedure on 172 healthy subjects are reported.

The use of detergents and immunoperoxidase reagents for the ultrastructural demonstration of internal immunoglobulin in lymph cells

•Lymph-borne immunoblasts were fixed in dilute glutaraldehyde and then treated with saponin. This treatment made most parts of the cells permeable to ferritin, so that anti-immunoglobulin (Ig) antibodies which had been conjugated to horse radish peroxidase (HRP) had no difficulty in gaining access to Ig which thus could be demonstrated at an ultrastructural level.•Best results were obtained by fixing the cells in 0.1% glutaraldehyde for 7 min and then treating them with a 1% solution of saponin for 100 min at 55°C before exposing them to the Ig—HRP conjugate. The method yielded reproducible results and although it causes a small amount of ultrastructural damage, it may be of value in detecting a variety of intracellular antigens.

Plasmodium berghei: High pressure liquid chromatographic analysis of nucleotides from erythrocyte-free malarial parasites

Isolation and analysis of the nucleotides from the malarial parasite, Plasmodium berghei, are described. This was accomplished by reducing contaminating leukocytes and platelets by settling in 6% dextran-Krebs glucose. Contaminating red cells (either parasitized or nonparasitized) were lysed by a technique using a dilute saponin solution in a Krebs-glucose medium. The parasites were collected by centrifugation and destroyed by addition of 10% trichloroacetic acid. The acid was extracted with Alamine-Freon TF and the nucleotides analyzed using high pressure liquid chromatography with microparticle anion exchange columns. The following compounds were separated and quantitated: ATP, GTP, CTP, UTP, ADP, GDP, CDP, UDP, AMP, GMP, CMP, UMP, and NAD. In nanomoles of nucleoside triphosphate isolated, the order of highest to lowest concentration was: ATP > UTP > GTP > CTP, while the order of diphosphates was: ADP > CDP > UDP > GDP, and the monophosphates: NAD > AMP > CMP > UMP > GMP. The order in amount of nucleotides present was: nucleoside triphosphates > nucleoside monophosphates > nucleoside diphosphates. The statistical analysis of the nucleoside triphosphates showed little deviation from sample to sample. A comparison of the sample from the host mouse red blood cell shows significant concentrations only of ATP, ADP, and NAD, indicating that contamination from host blood cells was unlikely.

Spectrophotometric method for the determination of total steroidal sapogenin.

A specific method for the spectrophotometric determination of total steroidal sapogenin, based on colour reactions with anisaldehyde, sulphuric acid and ethyl acetate and applicable to microgram amounts, is described.It has been shown that this determination can be carried out directly on a saponin solution and that there is virtually no interference from sugars, sterols, fatty acids and vegetable oil. The sapogenins have the same colorimetric properties whether they are in the free state, bound with sugars, esterified with acetic acid or mono- or polyhydroxylated. The method described is accurate (relative error 1.4%), rapid, easily automated and gives a chromophore with the same absorption spectrum with only one peak at 430 nm for all of the sapogenins tested: diosgenin, tigogenin, hecogenin, smilagenin, yonogenin, tokorogenin, etc. The molar absorption coefficient is approximately 49 000.

Vaccination of Pigs Against Hog Cholera (Classical Swine Fever) With a Detergent Split Vaccine

Four pigs were inoculated subcutaneously with a detergent (triton X 100) split hog cholera virus in Freund’s incomplete adjuvant. Four other pigs were in the same way inoculated with a detergent split bovine viral diarrhoea virus, also in Freund’s incomplete adjuvant. In the experiment were used 3 control pigs. The vaccinations were repeated after 3 weeks. All pigs were challenged with highly virulent hog cholera virus (Tubingen) 12 weeks after primary inoculations. Signs of hog cholera were only noted in the control pigs. This introductory experiment was succeeded by a larger experiment with subcutaneous inoculations of: 10 pigs with detergent split hog cholera virus in Freund’s incomplete adjuvant, 10 pigs with detergent split hog cholera virus in a saponin (Quil A) solution, 10 pigs with detergent split bovine viral diarrhoea virus in Freund’s incomplete adjuvant, 10 pigs with detergent split bovine viral diarrhoea virus in the Quil A solution plus 5 control pigs. The vaccinations were repeated after 3 weeks, and finally all pigs were challenged 9 weeks later with the highly virulent hog cholera virus strain. With the exception of 1 animal which died accidentally, all animals survived in the groups inoculated with the Quil A vaccines and in the group inoculated with the detergent split hog cholera virus/oil adjuvant vaccine. In the group inoculated with the detergent split bovine viral diarrhoea virus/oil adjuvant vaccine, some of the pigs died of hog cholera.

Changes in the Anatomy of Cotton Seed Coats Caused by Lucerne Saponins

The anatomy of the brown coat and white "membrane" of cotton seeds has been studied in relation to the inhibition of germination caused by lucerne saponins. Cationic, anionic, and nonionic surfactants were found to have an effect similar to that of saponins: they inhibited cotton seed germination and decreased the diffusion of oxygen through the seed coats but did not affect the embryos. Observations carried out with the aid of the light microscope and the scanning electron microscope showed that immersion of cotton seeds in the saponin and other surfactant solutions caused more swelling of the fringe and living cell walls of the "membrane" than immersion in water. The relation between this swelling and the inhibition of oxygen permeability through the "membrane" is discussed.

Citrate-saponin-acid method for the recovery of microfilariae from blood.

The combination citrate-saponin-acid procedure with its internal modifications of each of the separate standard methods, viz. , citrate, saponin, and Knott's, was developed to produce greater yields of microfilariae from equivalent amounts of peripheral blood of infected patients, especially those with scanty numbers of organisms, than the total yields recoverable by the three standard methods separately. Packed erythrocytes from 10.0 ml. of citrate-treated whole blood were destroyed through the action of a 0.5% saponin solution, after which centrifugation facilitated the recovery under low power magnification of motile microfilariae in wet mounts made from the sediment. Preparations designated mini-Knott's were made as wet mounts from two drops of sediment and were first examined for motile microfilariae and subsequently mixed in place with several drops of 1.0% acetic acid solution before drying and staining. Examination of these slides permitted the demonstration of concentrated numbers of straightened microfilariae under low power and identification under oil immersion magnification. Eighteen cases are reported.

Flow characteristics of horizontally moving stable aqueous foams

AbstractAn empirical study was made of the flow characteristics of a stable aqueous foam (based on dilute solutions of saponin) moving horizontally inside a duct. Characteristic foam data have been determined experimentally, namely, drainage, interfacial areas, and related dimensions such as lamella thickness and foam cell size, fractional liquid holdups, foam velocities, and extent of gas channeling through the foam. Simple correlatability of drainage, interfacial area, and fractional liquid holdup in terms of basic parameters such as the liquid flow rate entering the apparatus, and the longitudinal position in the duct has been demonstrated. The sometimes simple dependence on the geometry of the perforated gas‐liquid contactor and on the physical properties of the foaming solutions (surface tension and bulk viscosity) has also been described.

Experimental studies on the ciliary activity in the trachea

Recently, air pollution has become an increasing problem for the industrialized countries, since it is thought to be one of the causes of chronic bronchitis and bronchial carcinoma.The ciliated epithelium of the respiratory tract plays a very important role for the elimination and resorption of inhaled harmful materials. Many studies of cilia have been carried out both anatomically and physiologically. However, the mechanism of ciliary movement, the neural control of ciliary activity and the relationship between the ciliary activity and resorption activity are not well known yet. It is therefore the purpose of this study to elucidate above mentioned problems by means of a new method. The ciliary activity in the trachea in living rabbits was examined.The ciliary movements were observed by the microscope specially constructed for vertical light.They were observed as the variations in the light reflexes on the mucous membrane.These light reflexes were then transfered to the phototube where the variations in light intensity were changed to those in current intensity and these were recorded on an oscilloscope or an ink writer via an integrating circuit.The velocity of mucous flow was estimated by measuring the time when the India ink on the trachea mucosa moved 2mm distance.Following results were obtained:1) The rate of ciliary beat in the normal rabbit was 1140 beats/min. The velocity of mucous flow was 9.0mm/min.2) Both the ciliary movement and mucous flow were hardly affected by the atmospheric temperature between 25a and 35a, however, a low relative humidity brought about remarkable reduction of the ciliary activity.3) The rate of ciliary beat did not change after electrical stimulation for the vagul nerve or injection of autonomimetic drugs. The velocity of mucous flow, however, increased slightly at the parasympathicotonic condition.4) The ciliary activity decreased with the exposure of 10p.p.m. sulphur dioxide for 20 days and the absorption of radioisotope (Nal131) from the lower respiratory mucosa was increased. With increasing of the concentration of SO2 gas, the ciliary activity tremendously decreaseed within a short time. 5) The ciliary activity reduced after the local administration of 0.5% nicotine solution or 1% 3-4 benzpyrene solution to the trachea mucosa.6) The ciliary activity increased after the local administration of 0.1% lysozyme solution or saponin solution to the trachea mucosa.

DETECTION OF NEOPLASTIC CELLS IN THE BLOOD: TECHNICAL METHODS

After a review of the methods proposed for detection of cancer cells in the blood, a method based on hemolisis and filtration is described. Hemolisis is obtained by adding a few drops of a solution of saponin to the blood sample. By centrifugration of the hemolised sample, a buffy coat is obtained, which contains the leukocytes residual to hemolisis and possible cancer cells. The buffy coat is resuspended in an isotonic saline solution with addition of formalin which is then filtered through Millipore filter membranes with pores of 5 μm diameter. The cells concentrated on the surface of the filter are then fixed and stained on the filter itself, which is dehydrated in alcohol, cleared by xilene and mounted in balsam. The filters acquire a perfect transparence that makes it possible to examine microscopically the cells on their surface.

A Paradichlorobenzene, Saponin, Iron Mordant Sequence in the Staining of Meiotic Chromosomes ofIpomea

For the meiotic study of Ipomea spp., flower buds were stripped of the calyx and corolla and soaked in saturated aqueous paradichlorobenzene at about 28° C for 3 hr, transferred to acetic-alcohol (1:3) for 6 hr, then into 1% saponin solution and left overnight. They were mordanted in 1:3 acetic-alcohol saturated with ferric oxide for 24 hr and stained in a mixture of 1% aceto-carmine and 2% aceto-orcein with 1 N HCl in the proportion of 9:9:1. The preparations were mounted in 1% aceto-carmine for temporary use and made permanent by dehydration through the n-butanol schedule. The pollen mother cells had clear cytoplasm with deeply stained chromosomes.

医薬品の吸収と排泄に関する研究 (第4報)

Blood level of chloramphenicol in rabbits was measured after oral administration of chloramphenicol crystals or chloramphenicol ester preparation. Estimation of the blood level was made in a following manner. After administration of chloramphenicol, 0.5cc. of blood is drawn, mixed with 0.1cc. of 3.8% sodium citrate, placed in a glass-stoppered test tube, and shaken with 0.2cc. of 1% saponin solution to effect complete hemolysis. To this mixture, 2cc. of the Clark-Lubs buffer (pH 7) and 3cc. of isoamyl acetate are added and the mixture is shaken for 10 minutes to effect extraction. This is centrifuged, 2cc. of the supernatant is mixed with 1cc. each of 3% isonicotinic acid hydrazide solution and 6% sodium hydroxide solution, and shaken in a thermostat of 30° for 30 minutes. The supernatant is decanted and absorbancy of the colored underlayer is measured (filter, S-40; light path, 5mm.). The blood drawn before administration of chloramphenicol is treated in the same manner and its absorbance is measured as the control. Blood level of chloramphenicol is calculated from the calibration curve.