Schneider Method Research Articles

A Rapid Analysis of the Deoxyriboside Constituents in RNA and DNA Fractions Extracted by the Schmidt, Thanhauser and Schneider Method

A Rapid Analysis of the Deoxyriboside Constituents in RNA and DNA Fractions Extracted by the Schmidt, Thanhauser and Schneider Method

鼠癩レプローマの燐酸代謝

The studies were carried out in regard to the phosphorous metabolism of leproma.After intravenous injection of 32P-orthophosphate, rats were killed at serial intervals and the subcutaneous leproma were resected. The rate of incorporation of 32P into leproma was considerably rapid and increased consistently for the first 60 minutes, and relatively high levels of 32P were maintained following 24 hours. The murine leprosy bacilli were collected. The 32P was taken up by the bacilli somewhat delayed compared with leproma, but a parallel relationship was found between them.The phosphrous fractions of leproma resected at serial intervals one hour after injection of 32P were obtained by the Schneider's method. The 32P was rapidly taken up by the acid-soluble fraction and the specific activity increased consistently for one hour. The incorporation of 32P into the acid-insoluble fraction was significantly high in the phospholipid fraction in contrast to the low levels in the nucleic acid and phosphoprotein ones. The turn over rate of the phosphorous, however, was more active in the nucleic acid fraction than in others.

The extraction and estimation of nucleotides and nucleic acids from plant material

The methods available for the extraction and estimation of nucleotides and nucleic acids have been applied to a variety of plant tissues. It has been shown that, with the tissues investigated, either aqueous ethanol or dilute perchloric acid could be used to extract the soluble nucleotides. Components other than nucleotides contributed to the 260 μu optical density of the extract, and with most tissues only 20 to 30 per cent of the optical density was due to nucleotide material. Although purification of the nucleotides in the extract was attempted by various procedures, such as precipitation of the nucleotides as insoluble salts, extraction of pigments with organic solvents and ion-exchange treatments, no suitable general method was found. The separation into individual nucleotides by ion-exchange chromatography was the only reliable way of estimating the nucleotide content of a tissue. The Schmidt and Thannhauser procedure was the only general method suitable for the extraction of nucleic acids from plant tissue. Purification of the RNA mononucleotides before estimation was essential, but the anion-exchange treatment described by Smillie and Krotkov did not remove all the material which interfered with the estimation of the RNA by phosphate or ribose content. The separation of the mononucleotides by ion-exchange chromatography provided the most reliable method of estimation. The DNA content of the tissue as estimated by the Schmidt and Thannhauser method was considerably lower than that obtained by the direct, hot acid extraction of Schneider, due in part to an incomplete precipitation of the DNA after alkaline hydrolysis. The Schneider method was suitable for the estimation of RNA under special conditions, such as in tissue homogenates containing no cell debris or nuclei, while the Ogur and Rosen procedure appeared to be more useful as a method of RNA fractionation than for the quantitative measurement of RNA.

Factors affecting the Schneider procedure for estimation of nucleic acids

Discrepancies found in certain tissues between the amounts of DNA found by the Schmidt-Thannhauser and Schneider methods, led to a re-examination of the latter procedure. Losses of nucleic acids can occur during the initial washing with cold acid, especially with perchloric acid, if contact is prolonged, but claims that considerable loss of RNA can occur during lipid extraction were not substantiated. The Schneider method of extracting nucleic acids from the tissue residue with hot acid has been found to give variable results depending on the concentration of extractant and the temperature used. Mild conditions may fail to extract all the DNA, while more vigorous extraction can result in destruction of deoxypentose and also in extraction of chromogenic material interfering with RNA determination. There is some evidence that the degree of extraction of DNA may vary, not only with the tissue, but also with the species, and poor extractability appears to be confined to whole tissue since ectraction was satisfactory with isolated nuclei. A claim that the amount of blood in the tissue can markedly affect the extractability of DNA has not been confirmed.

Factors affecting the Schneider procedure for estimation of nucleic acids

Discrepancies found in certain tissues between the amounts of DNA found by the Schmidt-Thannhauser and Schneider methods, led to a re-examination of the latter procedure. Losses of nucleic acids can occur during the initial washing with cold acid, especially with perchloric acid, if contact is prolonged, but claims that considerable loss of RNA can occur during lipid extraction were not substantiated. The Schneider method of extracting nucleic acids from the tissue residue with hot acid has been found to give variable results depending on the concentration of extractant and the temperature used. Mild conditions may fail to extract all the DNA, while more vigorous extraction can result in destruction of deoxypentose and also in extraction of chromogenic material interfering with RNA determination. There is some evidence that the degree of extraction of DNA may vary, not only with the tissue, but also with the species, and poor extractability appears to be confined to whole tissue since ectraction was satisfactory with isolated nuclei. A claim that the amount of blood in the tissue can markedly affect the extractability of DNA has not been confirmed.

EFFECT OF X-RAY ON NUCLEIC ACID OF YOSHIDA'S SARCOMA

Sinec 1942, when Caspersson and Brachet published their studies, it hes become clear that nucleic acid is an important constituent of the cell, playing a primary part in its existence and development. There are, however, not many studies on alteration in nucleic acid amount in a short time after irradiation of the tissue or the cell, and on relation between this alteration and cell division. ln view of this, the author attempted by chemical method to determine nucleic acid concentration per cell of Yoshida's sarcoma, and to seek, from its change, the effect of irradiation on the cell.At 4-7 days after transplanting the sarcoma into rats, they were abdominally irradiated in single doses of 100, 200, 500 and 10, 000γ. After a definite time, ascite was taken, from which nucleic acid was extracted by Schneider's method. Then DNA and RNA per cell were colorimetrically detereined by diphenylamine reaction and orcin HCl reaction, respectively. The results were as follows : 1) Normal Yoshida's sarcoma gave relatively consant values, DNA per cell being 10.65± 0. 45× 10-6γ, and RNA 17. 98±1. 16 × 10-6γ.2) After irradiation with 100γ, both DNA and RNA showed a slight increase at 1 hour, but at 3 hours they returned to the initial values, exhibiting thereafter no remarkable change.3) after irradiation with 200γ, no change appeared as late as 3 hours, but appreciable increase at 6 hours, more reme remarkably in RNA than in DNA. The values transiently at 12 hours, but again rose at 18 hours, returning to the initial level at 24 hours.4) After irradiation with 500γ, DNA showed no change, while RNA gave slight increase at 18 hours to 24 hours.5) After irradiation with 10, 000γ, neither DNA nor RNA showed any definite tendency of change.6) A consistent relation could be seen between change in nucleic acid and number of cell division. After irradiation with 100γ, cell division decreased to the minimum level at 1 hour, rising abruptly thereafter, in correspondence with increase in nucleic acid. The same relation was observed at 6 hours after irradiation with 200γ, but no consistent relation was obtained after irradiation with 500γ.

Antibiotics as protectant fungicides against rice blast

Effect of an antibiotic, blasticidin S, on respiration of spores, mycelial pellets, and isolated mitochondria of Piricularia oryzae was studied. Blasticidin S was previously reported to be effective as a therapeutic fungicide against the rice blast.The spores were obtained by culturing the fungus on a medium consisted of 1% soluble starch, 0.2% yeast extract powder, 10% rice stem juice, and 1.5% agar, at 27°C for 10-14 days. The mycelial pellets were prepared by seeding 70ml of liquid medium (2% glucose, yeast extract powder 0.5%, rice stem juice 10%) in 500ml flasks with appropriate amount of spores, and culturing in a reciprocal shaking machine operating at about 120 strokes per minute at 27°C, for 3 days. The mitochondrial fraction was prepared from the above mentioned mycelial pellets by the Schneider's method. The respiration was measured in the usual way in a 12-unit rotary Warburg respirometer at 30°C.Blasticidin S partially inhibited the oxygen uptake of the spores in the presence of glucose, pyruvate, succinate, or glutamate at the minimum spore germination inhibitory concentration (1μg/ml). The inhibition was still not complete at a concentration of 10μm/ml. Endogenous respiratin of the spores was not inhibited at this concentration.Inhibition of respiration of the mycelial pellets caused by blasticidin S was almost the same as in the case of the spores. The oxygen uptake of the mycelial pellets in the presence of the abovementioned substrates was partially inhibited at the minimum mycelial growth inhibitory concentration (0.1μg/ml). Exogenous respiration was not completely inhibited at a concentration of 100μg/ml. Endogenous respiration of the pellets was not inhibited at this concentration.The oxygen uptake of the mitochondrial fraction of the mycelial pellets in the presence of succinate was inhibited by blastmycin (antimycin A) at a concentration of 5μg/ml, but not by blasticidin S at a concentration of 500μg/ml. Therefore, blastcidin S was not considered to be directly inhibiting the succinoxidase system in the fungus.

Purification and measurement of microgram amounts of radioactive nucleic acids and proteins from animal cells in tissue culture

Rapid procedures are presented for analyzing microgram quantities of animal cells which have incorporated either radioactive adenine, glycine, or phosphate during growth in tissue culture. After proteins, ribonucleic acid and deoxyribonucleic acid are separated using abbreviated versions of the Schmidt-Thannhauser and Schneider methods, the acid-precipitable fractions are collected on membrane filters. The acid-soluble nucleic acid derivatives are adsorbed selectively on charcoal, and this in turn is collected on a filter. Both types of filter are then counted directly. The radioactivity bound by charcoal was shown to be relatively free of the non-nucleic acid impurities, which commonly interfere with conventional analytical procedures.

THE ESTIMATION OF NUCLEIC ACIDS IN SOME ALGAE AND HIGHER PLANTS

Several current methods for the extraction and estimation of nucleic acids in biological materials were applied to Euglena and other plants. The efficiency of both the preliminary extractions for acid-soluble-P and lipid-P and the subsequent extraction of the nucleic acids was studied. A relatively high acid concentration (15% TCA) was required to directly extract all the acid-soluble phosphates. These conditions appeared to remove a small amount of the RNA. Lower acid concentrations as used in the Ogur–Rosen method (2% PCA) failed to extract all the acid-soluble phosphates. By using a modification of the Ogur–Rosen initial extraction method, the acid-soluble phosphates were quantitatively extracted without loss of RNA. After removal of the acid-soluble phosphates and lipid phosphates, the plant nucleic acids were quantitatively extracted by either the Schmidt–Thannhauser or Schneider methods. In many of the plants tested, the presence of pentose-containing polysaccharides, protein degradation products, or polyphosphate (algae only) interfered in estimations based on either the Schneider or Schmidt–Thannhauser procedures. Such interfering substances in the Schmidt–Thannhauser method were eliminated by the use of an anion exchange resin. Details are given of a modified Schmidt–Thannhauser procedure which should be suitable for a wide range of plants. The modified procedure may be simplified for Euglena and some higher plant tissues depending on the nature and quantities of interfering substances present. Methods are also given for the quantitative separation of plant RNA nucleotides by paper chromatography and by ion exchange paper chromatography.

SERUM TRANSAMINASE ACTIVITIES

The author's colorimetric assay method of serum transaminase (T) and its modification for tissue T were used in these studies. The results were indicated as γ of oxalacetic acid or pyruvic acid, which was formed after the incubation time under definite conditions. RESULTS A. Clinical Studies: 1. Normal values of serum T were as follows: The mean of serum glutamic-oxalacetic transaminase (SGOT) was 8.10±2.15γ (180 cases) and that of serum glutamic-pyruvic transaminase (SGPT) was 4.10±1.63 γ (120 cases). 2. SGOT was elevated but SGPT was unchanged in a case of acute myocardial infarction. In various kinds of liver diseases, especially in acute hepatitis, both were markedly elevated. 3. The clinical course and examination data of six representative cases were described and briefly discussed. 4. The difference of mobility between SGOT and SGPT was clinically and experimentally established from paper-electrophoretical studies. B. Experimental Studies: 1. Not only an increase of serum T activities but a decrease of liver T activities was found in CC14 treated mice. With these findings and the histological examination of the liver, it was confirmed that serum T activities was a good indicator of acute liver damage. 2. Localization of T in cellular fractions of liver homogenate was studied by Schneider's method. The decrease of T activities was most remarkable in the mitochondrial fraction, in CCI4 treated rabbits. 3. The increase of serum T activities was compared with the decrease of liver T activities in CC14 treated rabbits. In this study, total amount of increased serum T activities was calculated from the serum T activities per 0.1cc and the circulating plasma volume which was determined by radioactive phosphorus method. The ratio of increased SGOT to decreased liver GOT was 17.3% and it was 41% in GPT. Dr. Wroblewski's presentation, in which SGPT was clinically more useful than in SGOT in the detection of acute liver damage, was experimentally confirmed with these data.

植物のウイルス感染機作 (II)

The work reported here mainly concerns the amounts of phosphorus compounds in various fractions of the tobacco or tomato leaves during the course of tobacco mosaic virus-infection. The homogenized juices of leaf tissue were fractionated by Schneider's method and inorganic phosphate liberated was determined by Allen's method using electrophotometer. Most noticeable effect of virus infection on phosphorus compounds was on nucleic acid fraction either from the experiments on detached leaf placed in nutrient solution or those on plants grown in pots. Usually 20-50 per cent increased amounts of them, especially of ribonucleic acid, were demonstrated in detached leaf 24-48 hours after inoculation. Essentially the same results ware obtained in the upper leaves when the lower leaves of the same plants were inoculated with the virus.By way of the same fractionation procedure of leaf-homogenized juices, it happens that the increased RNA content was, in one case, observed in the supernatant fraction after centrifuging ×20, 000g, or, in another, in the precipitates by 100g centrifugation. No attempt was made to solve this problem. P32 in nutrient solution in which the inoculated leaves were floated, was found to incorporate into virus nucleic acrd 3 days after inoculation. Apart from this, the locally respond leaves of Nicotiana glutinosa showed no increased amount of nucleic acid when they are inoculated with TMV.The amounts of acid soluble phosphorus compounds, especially inorganic P, was higher in the inoculated leaf than in the sound. The difference in organic P content betwen the sound and infected leaves was still inconclusive, although it seemed likely to decrease in the latter. Further, the protein and lipid of the infected tobacco leaf comprised much amount of P than those of the sound throughout the infection. At our present level of understanding, it seems more reasonable to emphasize the fact, from these findings, that no evidence about a shift of the metabolic process of leaves incited by viruses could be demonstrated except the increased amount of virus nucleic acid.

THE PHOSPHORUS FRACTIONS OF BACILLUS CEREUS AND BACILLUS MEGATERIUM: I. A COMPARISON OF SPORES AND VEGETATIVE CELLS

The phosphorus fractions of the spores and young vegetative cells of B. cereus and B. megaterium were compared using the methods of Schneider and of Schmidt and Thannhauser. Differences were found, not only between the two types of cell, but also between the species studied. The spores of B. cereus and B. megaterium contained, on a dry weight basis, similar amounts of nucleic acid, yet the spores of B. megaterium contained twice as much total phosphorus as those of B. cereus. The excess phosphorus of B. megaterium spores was present as an acid and alkali insoluble residue and was made up of empty spore coats. While this same fraction accounted for only 4% of the total phosphorus of B. cereus spores it made up some 60% of that of B. megaterium. A similar fraction from vegetative cells was low in phosphorus. The spore coats of B. megaterium, in contrast to the cell walls were lysozyme-resistant. Cold acid-soluble and lipid phosphorus could be properly estimated only on disrupted spores. Disruption also proved essential for the ready extraction of spore nucleic acids with hot TCA, but did not greatly alter the solubility of the residue phosphorus. Ribosenucleic acid (RNA) comprised 50% of the total phosphorus in the spores of B. cereus (3–4% of the dry weight) but only 25% in B. megaterium spores. In both species the RNA of the vegetative forms accounted for a larger proportion of the total P of the cell. Ribonuclease digested the RNA of spores and vegetative cells to the same degree. The Schmidt and Thannhauser method was found more suitable than the Schneider method for the estimation of desoxyribosenucleic acid (DNA). The DNA content of B. cereus spores was about 1% of their dry weight; that of B. megaterium spores was slightly less. Some 12–20% of the phosphorus of B. cereus spores and 6% of that of the young vegetative cells was present as acid-labile (non-nucleic acid) phosphorus which exhibited some of the characteristics of polymetaphosphate.

P32による実験的日本脳炎に関する研究

In the previous paper by the same author the nucleic acid metabolism in some organs which was infected by Japanese B Encephalitis was studied by Schneider's method. Some detailed study by Schmidt & Thanhauser's method on this problem was further undertook and is going to be reported here.P32 was used as the tracer throughout this investigation and was injected intraperitoneally into the mice at the acme stage of infection.After the time intervals of 6 and 24 hours were collected certain quantities of brain, which were then fractionated by Schmidt & Thanhauser's method into DNH- and RNA- fractionsThese nucleic acid fractions were investigated on their P32 contents.By comparing the results with those obtained from the control references made by treating the healthy mice with P32 in the same way, an increase in P32-activity was recognized at the DNA-fraction of the brain, this indicating the fact that, when mouse is infected with the virus, the DNA-fraction of the brain suffers a marked change.Similar investigation was also carried out at the incubation period.In this case, however, an increase in the P32-activity was observed at the DNA and PNA-fraction of the liver collected from the mice infected with the virus. It is fully interesting that this increase in P32-activity shows intimate accordance with the visceral phase of the disease.

A multiple-unit plastic container for processing unmounted sections.

The use of polyethylene containers eliminates many disadvantages of those made of glass. A container consisting of 35 individual units (baskets) mounted vertically in a polyethylene disk 43½4 inches in diameter and 1½8 inch thick was a very successful substitute for glass baskets when used in Schneider's (1952) shaker-staining method. Each basket was made from a 2-inch length of 56-inch outside diameter and ½-inch inside diameter polyethylene tubing, with its bottom end closed by a fused-in circular piece of 100-mesh nickel screen. The baskets were inserted through tight holes bored in the disk by a No. 10 cork borer. The multiple-unit assembly was fitted with a handle made of polyethylene rods. Staining was the same as that obtained in glass baskets and polyethylene was found to resist the usual histological reagents at temperatures below 70 °C.

Growth and metabolic activities of developing wings and legs of the chick embryo: I. Establishment of multiplicative growth rates on the basis of desoxyribosenucleic acid

1. 1. The multiplicative growth rates of wings and legs of 5- to 12-day chick embryos were calculated on the basis of desoxyribosenucleic acid (DNA) which was extracted according to Schneider's method and estimated with Dische's diphenylamine reagent. 2. 2. The overall average and range of DNA content per million nuclei were the same for the legs and the wings. 3. 3. On a semi-logarithmic scale, the increases in DNA and wet weight showed a linear trend for both appendages. 4. 4. On the 5th day of incubation, the mean DNA content per wing was not significantly different from that of a leg. During further development, the legs accumulated DNA at an appreciably higher rate than the wings. The instantaneous rate of DNA increase were calculated. The ratio of these rates (leg/wing) was highest at the 6 to 7 day interval. 5. 5. During the 5–8 day periods, the times required for the quantity of DNA per leg to double were between 25 to 27 hours; those per wing, between 32 to 39 hours. 6. 6. The rates of increase of DNA (cell number) were compared with those of wet weight (overall growth) and were found to be lower. 7. 7. The unit DNA as an expression of a given cell population was suggested as a reference for metabolic studies.

Nucleic acid content in relation to cell size in the mature larval salivary gland of Drosophila melanogaster

1. 1. The content of desoxypentose (DNA) and pentose (PNA) nucleic acids was determined in mature larval salivary glands of Drosophila melanogaster. Use of a stock giving giant and normal-sized larvae permitted the quantities (expressed on a per cell basis) to be related to different cell sizes. 2. 2. A micromodification of the Schneider method was developed which allows determination of samples consisting of 18 pairs of salivary glands (approximately 5400 cells) containing a total of 10–20 μg. N, 1–2 μg. DNA, and 10–20 μg. PNA. Data from some experiments using perchloric acid as the extraction medium are also included. Details of the extraction procedure and comparison of the nucleic acid values obtained from sugar determinations, phosphorus, and ultraviolet absorption are given. A discussion of the validity of the measurements is also included. 3. 3. The results obtained show that there is an increase of PNA per cell with doubling of cell size but no measurable increase of DNA. The increase in the PNA content is, however, not as great as that previously (1) found for nitrogen and alanylglycine peptidase in comparable material. 4. 4. The absolute values of DNA (284 μμg.) and PNA (2250–3600 μμg.) found per cell are compared with those found by others in related types and sizes of cells. In addition, the changes in DNA and PNA with cell size are correlated with similar measurements made elsewhere on materials undergoing various types of growth.

ヴィールス感染マウス臓器内における放射性同位元素P<sup>32</sup>の分布

Many mice were inoculated with virus (the mouse-adapted Mochizuki-strain dengus virus) intracerebrally. At various times after the inoculation, 8-10 mice were picked up at random and were injected with P32-solution intraperitoneally, then half of the mice (4-5) were sacrificed 6 hours, and the other half 24 hours, respectively, after the P-administration, by bleeding through carotis-arteriae. Of each group, the brains were removed and pooled, and the phosphorus compounds contained were separated into fractions of acid-soluble phosphorus, phospholipid, phosphoprotein and nucleic acids, by Schneider's method. Each fraction thus obtained was subjected to the phosphorus-determination (by Allen's method) as well as radioactivity-measurement (with the GeigerMüller tube). As a control, the same tests were conducted with the materials from mice injected with a normal-mouse-brain-emulsion intracerebrally. Results obtained were as follows: (1) No definite differences were evidenced between the total phosphorus quantity of each group. (2) The radioactivities of the acidsoluble phosphorus fractions were higher in the infected groups than in the control groups. The significance was already apparent during the early stage of the murine infection, and became more distinguished with the progress of time. (3) The radioactivities of the phosphoprotein and nucleic acids fractions increased during the acme stage of the infection, while those of the control groups remained almost constant. It was concluded from these results that the permeability of the blood-liquor-barrier, as well as the metabolism centering around phosphoprotein and nucleic acids, rose distinguishedly in the organs of the virus-infected mice.

Neurocirculatory Reactions in the Psychoneuroses Studied by the Schneider Method. (Amer. Journ. Psychiat., vol. cxiii, p. 567, Nov., 1936.) McFarland, R. A., and Huddleson, J. H.

<i>Neurocirculatory Reactions in the Psychoneuroses Studied by the Schneider Method</i>. (<i>Amer. Journ. Psychiat., vol. cxiii, p</i>. 567, <i>Nov</i>., 1936.) <i>McFarland, R. A., and Huddleson, J. H.</i>