Fraction B2 Research Articles

Chemical and biological properties of bleomycin labelled with 57Co.

The chemical composition of bleomycin labelled with 57Co and its distribution and kinetics in the organs of tumour bearing rats were studied. For separating bleomycin, the chromatography on a CM-Sephadex C-25 column with a gradient of ammonium formate was found to be most suitable. It is also possible to use paper chromatography in 10% NH4Cl and thin layer chromatography in 10% ammonium acetate with methyl alcohol 1:1. The fractions obtained by the chromatographic separation were identified as A2, B2 and A1. Labelling with 57Co given rise to a more complicated mixture of labelled compounds, all of them differing chromatographically from the original non-labelled components. Using carrier-free 57Co two main radioactive compounds are formed apparently corresponding to the fractions A2 and B2 containing one atom of Co per molecule. The amount of another two labelled components grows progressively with increasing amount of carrier CoCl2. Since their chromatographic properties are even more different from those of the original compounds, it was concluded that they probably contain two atoms of Co in their molecules. During distribution studies, rapid excretion of labelled bleomycin into the urine and stool was observed. Increased activities were retained in the liver, kidney, intestine and tumour.

AN IN-VITRO IMMUNOLOGICAL ASSAY FOR DIAGNOSIS OF CŒLIAC DISEASE

Production of leucocyte-migration-inhibition factor (L.I.F.) by peripheral-blood lymphocytes in response to challenge with gluten fractions was studied in 55 patients with cœliac disease and in 32 controls. 96% of the patients with cœliac disease demonstrated significant L.I.F. reaction in response to gluten fractions irrespective of their dietary status. Only 2 out of 32 controls had a positive reaction. This was in response to the B2 or B3 fraction, but never to both. The agarose microdroplet method of L.I.F. assay is reliable and technically simple enough for use in most clinical laboratories. The assay of L.I.F. production by peripheral-blood lymphocytes in response to gluten fractions, would be a useful adjunct in the diagnosis of gluten-sensitive enteropathy.

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50–500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantity of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by the electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-–0.3 μm long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of ε-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.

Effect of aflatoxin on complement activity in guinea pigs.

SummaryGuinea pigs were dosed once daily with partially purifed aflatoxin in measured amounts per os for 20 days. A significant average depression of complement titers occurred in guinea pigs given a daily dose of aflatoxin equivalent to 0.03 mg or greater of fraction B1. Guinea pigs given a daily dose of aflatoxin equivalent to 0.01 5 mg or greater of fraction B1 had average weight significantly below that of control guinea pigs. Microscopic changes in the liver were detected mostly in guinea pigs given 0.03 mg or more of B1 equivalents. Considerable individual variation in complement titers, weights, and liver changes were noted among the guinea pigs.

Changes in the proportions of two mitochondrial populations during the development of embryonic chick liver.

1. Two populations of morphologically intact mitochondria were isolated from embryonic, neonatal and adult chick liver by isopycnic centrifugation. 2. The protein/phospholipid ratio of the total mitochondrial fraction, the low-density mitochondria (B2, d1.176) and the high-density mitochondria (B3, d1.206) did not differ significantly. 3. During development there is a marked increase in the B2 fraction in relation to the B3 fraction. 4. Cytochrome oxidase and malate dehydrogenase activities as well as respiratory control increased during the embryonic development of the chick, though their rates of increase were not correlated. 5. In the three different embryonic stages that were investigated, as well as in the neonatal and adult chick, the protein/lipid as well as the protein/phospholipid ratio stayed constant and showed no progressive increase, as had been previously reported. 6. It was shown that forces greater than 18400g(av.) for 2h have to be used before chick liver mitochondria reach isopycnic equilibrium. 7. As for rat liver mitochondria, the constant protein/phospholipid ratio of the B2 and B3 fractions and their apparent morphological intactness leads one to conclude that the matrix space of B2 mitochondria is inaccessible to sucrose, whereas B3 mitochondria possess an inner membrane that is permeable to sucrose.

Metabolic vitamin B complex deficiency due to the administration of glucocorticoid.

The influence of continuous use of glucocorticoid on the metabolism of vitamin B complex, especially B2, PaA and CoA in young children and rats was investigated. The following results were obtained.1. The observation upon the B2 metabolism in the children receiving continuously large amounts of the hormone shows that the blood B2 content and urinary B2 excretion after loading an adequate amount of FR are higher than in control children not given glucocorticoid. These changes are supposed to be reversible.2. Blood B2 and tissues B1, B2, PaA and CoA contents are increased in the early stage and decreased in the late stage by the continuous use of the hormone in rats. The changes in FAD fraction or CoA content are remarkably distinct compared with other B2 fractions or PaA.3. In adrenalectomized rat liver the total B2 content is slightly decreased, but the FAD fraction is reduced markedly. And also, it is proved that the FAD synthetic activity in adrenalectomized rat liver is definitly interfered with and again, CoA content in such liver is significantly suppressed.4. From these results the possibility for the occurrence of the iatrogenic disturbances in vitamin metabolism by the administration of large amounts of the hormone is suspected.

MOENOMYCIN. VII

The new antibiotic, moenomycin, isolated from Streptomyces bambergiensis is a mixture of several, chemically very similar components, which are particularly characterized by their phosphorus content. The moenomycin complex can be separated into components A, B1, B2 and C by means of column chromatography on silica gel with n-propanol - 2N ammonia as eluant. The use of other chromatographic procedures, e. g. chromatography on anion exchange resins, results in a further separation of fraction B1 into components D, E and F ; similarly fraction B2 can be separated into components G and H. Isolation and properties of moenomycins D, E, F, G and H are described.

Antineoplastic Activity of Aflatoxin B1

THE aflatoxins, a group of related mycotoxins produced by Aspergillus flavus, have been extensively evaluated for biological activity. They are carcinogenic in animals1–5. The aflatoxin B1 fraction has been teratogenic in hamsters6, probably mutagenic in micro-organisms7 and caused chromosome breakage in mammalian cell cultures7,8. The biological activity of aflatoxin B1 was similar to that of known alkylating agents7. This investigation was undertaken to determine whether it had antineoplastic activity.

Mycotoxins: Aflatoxin Isolated from Penicillium puberulum

Penicillium puberulum Bainer was found growing on a sample of moldy peanuts. It also grows on shredded wheat, potatoes, and laboratory culture media such as wort, potato dextrose, and Sabouraud agars, and synthesizes aflatoxin on these substrates. Thin-layer chromatograms of the chloroform-soluble toxin produced by the mold when grown on shredded wheat show fluorescent bands with R F values identical with those of the fractions B 1 , B 2 , G 1 , and G 2 of the toxin produced by Aspergillus flavus . This extract produces typical bile duct proliferation type of liver damage in 2- to 3-day-old Peking white ducklings.

Electrophoresis of the blood of the silkworm, Bombyx mori on filter paper

1) The daily change of the electrophoretic pattern of the silkworm blood from larval to pupal stage was examined. Three components (b3, b2, b1) were present in the blood of the early period of the 5th instar as shown in the previous paper (AIZAWA, 1955), while from 4-5th day of the 5th instar to pupal stage, there appeared two components, which were densely stained (Figs. 1, 3). In the latter case, when the electrophoresis was performed with the diluted blood, three protein components could be clearly detected (Fig. 2). b3 is the fastest moving component and it seems to be albumin. b2 and b1 are probably globulin. b3 component decreased with the pupal age and finally disappeared, at earlier time in male than in female.2) Comparison of the electrophoretic patterns was made by veronal, phosphate, citrate, borate or tris (hydroxymethyl) aminomethane buffer with the varying ion concentration and pH. The separation of the protein fractions with veronal buffer (pH 8.6, I=0.05) was better than with other buffers.3) Comparison of staining, was examined using bromphenol blue, solar blue black and amido black. All dyes were suitable for the purpose. By sudan black staining, b3 component was slightly stained (Fig. 4).4) Paper strips were cut off from the electrophoresis paper of the jaundice-diseased blood at the regions of b3, b2+b1 and the starting line as shown in Fig. 5. The virus was extracted with the distilled water and the injection experiments were performed. The virus amount was highest in b2+b1 fractions and it decreased in the fraction from near the starting line. The virus activity was scarecely shown in the fraction of b3.5) Any difference, except for quantitative one was not observed in the electrophoretic patterns of the blood between the normal and diseased larvae (both nuclear and cytoplasmic polyhedroses). On the contrary, the pattern of Galleria mellonella is, however, different from that of Galleria-adapted silkworm jaundice virus (Aizawa, unpub.).

A Complex Polysaccharide Fraction from the Cells of the Human Type of Tubercle Bacillus, H 37.

The recent work of Avery and Goebel1 has shown the importance of omitting the use of alkali in the purification of the specific polysaccharide of Type I pneumococcus. It was accordingly deemed advisable to repeat the isolation of the tubercle bacillus fractions described in a recent note,2 omitting the steps involving precipitation from alkaline solution, and keeping all solutions at least at a slight acidity. While the difficulties of the fractionation were increased, the B1 and B2 (low rotating) fractions were ultimately isolated in the same form as before. However, the C fraction (high rotating, insoluble in 75% methyl alcohol) differed markedly in its chemical properties. Aqueous solutions of the substance were opalescent, even after passage through a Berkefeld filter, and, on treatment with alkali, slowly deposited a bulky precipitate consisting mainly of magnesium palmitate. Magnesium was identified as the double ammonium phosphate, while the palmitic acid, crystallized from 60% methyl alcohol, melt...

Specific and Non-Specific Cell Polysaccharides of the Human Type of Tubercle Bacillus, H37.

Six hundred gram portions of ground, defatted human type (H37) tubercle bacilli21 were percolated with dilute acetic acid containing 0.5% of phenol until the neutralized effluent no longer reacted strongly with antiserum. The solutions were concentrated to small bulk in vacuo and precipitated with several volumes of alcohol. Further fractionation involved precipitation with glacial acetic acid, with alkali and alcohol, removal of the glycogen with saliva, removal of material precipitable with barium hydroxide (phosphates and a fraction [A]), separation of the residue into 3 main portions, (B1) soluble in 85% methyl alcohol; (B2) soluble in 75-85% methyl alcohol, and (C) insoluble in 75% methyl alcohol. Each portion was further purified by fractionation with glacial acetic acid or by special procedures which will be described in a more detailed communication. Fractions B1 and B2 are perhaps identical. Properties of the fractions are given in Tables I and II.It is evident that at least two specific polysacc...