Humic-like substances of bacterial origin. II. Fractionation of the bacterial humic-like substances by gel filtration on sephadex gels.

Humic-like substances obtained from cells of Pseudomonas acidovorans were separated on Sephadex G-25 into two groups of substances of different molecular weight. The substances of the molecular weight greater than 5000 were successively separated on Sephadex gels G-50, G-75, G-100. Five fractions of different molecular weight were obtained, the percentage of which varied depending on the media used and time of incubation of the bacteria. Most (38%--46%) of the compounds contained in the bacterial humic acids were of approximate molecular weight of 40 000--50 000. The distribution of the fractions in the bacterial "humic-acids" was compared with those of the humic acid made by Fluka A. G. The synthetic humic acid contained most (approximately 40%) of the compounds of approximate molecular weight of 8000--10 000. In the bacterial and synthetic material the content of the compounds with the molecular weight above 100 000 was very similar (8%--12%).     

Characterization of a gonadal factor involved in the control of FSH secretion.

This communication presents evidence for the existence in the ovine testis of proteinaceous factors which suppress LH as well as FSH. Isolation of these factors has been achieved by using three different procedures: cytosol preparation, metaphosphoric acid extraction and ultrafiltration. Chromatography of cytosol or metaphosphoric acid extract on Sephadex G-75 resulted in separation into three protein fractions designated as G-75-I, II and III in order of their elution. When administered to castrated male rats, Fraction G-75-I suppressed circulatory levels of LH (53% inhibition, P less than 0.05) without altering FSH. The most retarded fraction, G-75-III, suppressed FSH (29% inhibition, P less than 0.001) without any concomitant change in LH. When fraction G-75-III was further fractionated on Sephadex G-25, three components were found and two, G-25-II and G-25-III, were biologically active. These fractions were homogeneous on polyacrylamide disc-gel electrophoresis. The FSH-suppressing factor (inhibin) was heat labile and susceptible to trypsin digestion, indicating that it is proteinaceous. Treatment with urea did not reveal any subunits. The molecular weight of this factor, as determined by gel filtration and SDS-urea gel electrophoresis was estimated to be around 1400-1500. The absence of sialic acid and the molecular weight data suggested that the isolated material was a simple protein and probably a small peptide. Gel filtration on Sephadex G-75 of the metaphosphoric acid extracts of liver, kidney, testis and ovary revealed an identical elution pattern for ovarian and testicular inhibin.     

Structural features of naegeli amylodextrin as indicated by enzymic degradation

Naegeli amylodextrin is the insoluble residue remaining after prolonged treatment of native starch granules with strong aqueous acid. The Naegeli amylodextrin from waxy maize starch was separated by gel chromatography on Sephadex G-50 into three fractions. Although the fractions were heterogeneous, their average structures were examined by enzymic degradation with porcine-pancreactic alpha amylase, beta-amylase, and pullulanase. The results show that Fraction I (highest molecular weight) has complex branching, Fraction II (major component, d.p. ～25) contains about one branch per molecule, and Fraction III (d.p. ～12) is mostly linear. Formation of these acid-resistant fractions may be explained as arising from a cluster model of amylopectin in which the outer chains are in a double-helical, crystalline arrangement.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Substances with alpha-melanotropin-like immunoreactivity in bovine brains.

1. Bovine cerebral hemispheres were extracted with an acidic medium (acetone-water-hydrochloric acid mixture, 40:5:1 by volume, pH 1.8). The precipitate which formed upon addition of a copious volume of cold acetone to the extract was designated acid acetone powder (AAP). 2. The AAP was then subjected to ion exchange chromatography on carboxymethyl (CM)-cellulose, gel filtration on Sephadex G100 and Sephadex G25, second ion exchange chromatography on CM-cellulose and high performance liquid chromatography. The absorbance of all fractions was measured at 280 nm and their alpha-melanotropin-(alpha-MSH)-like immunoreactivity was monitored with radioimmunoassay. 3. It was found that alpha-MSH-like immunoreactivity and bioactivity (lipolytic activity) was due to low molecular weight materials as evidenced by their retardation on Sephadex G-100 and Sephadex G-25. The immunoreactivity was distributed among fractions adsorbed and fractions unadsorbed on CM-cellulose and also among high performance liquid chromatographic fractions signifying the presence of multiple alpha-MSH-like molecules.     

Nicotinamide: suppression of lymphocyte transformation with a component identified in human transfer factor.

The component in human transfer factor (TF) (Fraction IV, from exclusion chromatography on Sephadex G-25) responsible for suppression of antigen-induced lymphocyte transformation was previously identified as nicotinamide. Commercially available nicotinamide was subsequently shown to produce suppression of antigen-induced responses in vitro previously observed with TF Fraction IV. Nicotinamide was found to be nontoxic at the highest concentrations employed (10(-2)M) and suppressive over a relatively broad range (10(-5) to 10(-2)M. The suppression appeared to be related to the magnitude of antigen- or mitogen-induced transformation and was apparent even when nicotinamide was added as late as 48 hr after stimulant addition.     

Structure of shigella antigens. Heterogeneity of specific polysaccharides of 2 Shigella flexneri strains and 2 Sh. flexneri/Escherichia coli hybrids]

The S-specific polysaccharide from 2 Sh. flexneri wild strains (with serological var. X- and var. Y-specificity, respectively) and 2 Sh. flexneri E. coli hybrids (with the same specificities) can be separated by means of gel chromatography on Sephadex G-200 and G-50 into altogether 6 fractions per strain. Fraction G-200/1 (molecular weight greater than 10(6)D) represents a polymer consisting nearly exclusively of glucose and is present mainly in the two Y-type strains, much less in the two X-type strains. Fractions G-200/2 and G-200/3 (molecular weight approximately 10(5)D and approximately 2 - 10(4)D, respectively) seem to consist mainly of the S-specific side chains while fraction G-50/2 (molecular weight approximately 2000 D) presumably contains an SR-polysaccharide (core with one repeating unit.) Fraction G-50/3 (molecular weight approximately 100 D) contains the core polysaccharide and fraction G-50/4 splitting products (mainly KDO). No significant differences in chromatographical behaviour and quantitative composition could be found between the polysaccharides of the wild strains and the hybrid strains. Because of the well-known stability of the glucosaminyl linkages the sugar analysis was not only performed after acidic hydrolysis. In some cases the acid hydrolysate was reacted with HNO2 to cleave the glucosaminyl linkages. In most cases the values obtaines now were higher than those obtained directly.     

SEPARATION OF NEUROTOXINS FROM HORNET (VESPA INSULARIS) VENOM AND THEIR ACTIONS ON CRUSTACEAN NEUROMUSCULAR TRANSMISSION

Abstract— The venom of homet ( Vespa insularis) was separated into three components by gel filtration on Sephadex G-50, Sephadex G-25 and Sephadex G-10. The effect of each component on crustacean neuromuscular junctions was studied electrophysiologically using intracellular recordings. Two components (Fractions D and E) suppressed the postsynaptic potentials; Fraction D preferentially depressed excitatory postsynaptic potentials (epsps) without affecting inhibitory postsynaptic potentials (ipsps), and fraction E depressed both epsps and ipsps with concurrent decrease in membrane resistance. Another component (fraction F) augmented both epsps and ipsps. Fractions D and F were possibly acting presynaptically while fraction E was acting on the postsynaptic membrane. The active substances in fraction D, E and F were separated on TLC and the active substance in fraction F was confirmed to be serotonin. The active substances in fractions D and E are assumed to be peptides from the results of peptidase digestion. The effect of fraction D was degraded by treatment with trypsin but not by chymotrypsin nor carboxypeptidase A and B. On the other hand, the effect of fraction E was degraded by chymotrypsin and carboxypeptidase B but not by trypsin nor carboxypeptidase A.     

SEPARATION OF TWO FACTORS AFFECTING THE AGGREGATION KINETICS FROM THE CONDITIONED MEDIUM

Two effective factors contained in medium "conditioned" with a large number of cultured cells were separated through Sephadex G-25 gel, by assaying the plating efficiency and growth rate of chicken embryonic cells in secondary culture. The first factor, which is contained in the heavy molecular weight fraction α, can promote plating efficiency of the inoculated cells at low density. The second is contained in the light molecular weight fraction β, and promotes the growth rate of sparsely cultured cells. The effects of these factors upon the aggregation kinetics of singly isolated cells were studied. The factor in the fraction α promotes aggregation to result in fast flocculation. The factor in the fraction β, while not effective by itself, affects the aggregation of the cells treated with the factor in α. In the co-presence of both α and β, a "lag" was found after the initial fast flocculation. The possible mechanisms of the interaction of these two factors on the cell surface are discussed.     

STUDIES ON PROTEIN METABOLISM IN HIGHER PLANTS IV. CHANGES IN PROTEIN COMPONENTS IN DETACHED LEAVES OF TOBACCO

Soluble protein fractions from tobacco leaves before and aftercuring were compared. The results of Sephadex G-200 or G-75chromatography and immunological experiments showed that theamount of larger molecular weight proteins diminished or greatlydecreased and that smaller molecular weight proteins accumulatedduring 3 days at 40 and 90% humidity after excision of theleaves from the stem. Fraction I, which was the largest proteinin the leaf extract and occupied about one-half of the solubleprotein before curing, was not found in the proteins after curing.On the contrary, the proteins contained in II-4 fraction, whichwere supposed to have smaller molecular weights, increased three-foldduring the curing. The origin of the smaller proteins was discussed. (Received May 11, 1967; )     

Effect of dialyzate fractions of spinach on growth of human-derived cells

Aqueous dialyzate of spinach was separated by Sephadex G-100, G-25 gel-filtrations and DEAE-cellulose ion exchange chromatography, and the effects of the fractions on growth of human-derived normal and cancer cell lines were studied. One of the fractions (SPW2) from a Sephadex G-100 gel-filtration of dialyzate promoted growth of a hybridoma cell line (HB4C5). Sephadex G-25 gel filtration of the SPW2 fraction produced four main fractions; SPW2-1, SPW2-2, SPW2-3 and SPW2-4. Among them, the SPW2-1, SPW2-3 and SPW2-4 fractions promoted the growth of a histiocytic lymphoma cell line (U-937) and hybridoma cell lines (HB4C5 and SI102). Both SPW2-3 and SPW2-4 fractions inhibited the growth of a breast cancer cell line (MCF-7). The SPW2-3 fraction, especially, was found to inhibit growth of cancer cell lines such as MCF-7, a differentiated hepatoma (HuH-7), a lung adenocarcinoma (PC-8), a lung squamous carcinoma (QG-56), and a lung anaplastic carcinoma (QG-90) more preferentially than that of normal cell lines. It was also found that a constituent of the SPW2-3 fraction caused the morphological alteration of U-937 cells in serum-free medium.     

Triwaglerin: a potent platelet aggregation inducer purified from Trimeresurus wagleri snake venom

Trimeresurus wagleri venom is the most potent inducer of platelet aggregation among the seven Trimeresurus snake venoms tested. By means of CM-Sephadex C-50 column chromatography, T. wagleri venom was separated into 19 fractions. Fraction XVI possessed the strongest aggregating activity and was further purified by Sephadex G-75 and on heparin-agarose columns, and finally Triwaglerin, with a molecular weight of 68000, was obtained. Its aggregating and ATP-releasing activity was dose-dependent and 10-times more potent than the crude venom. Triwaglerin was devoid of any of the enzymatic activities possessed by the crude venom. Triwaglerin-induced aggregation was not affected by indomethacin, creatine phosphate/creatine phosphokinase (CP/CPK), platelet-activating factor (PAF) antagonists, verapamil or heparin, but was inhibited completely by mepacrine, imipramine and forskolin and markedly by tetracaine and sodium nitroprusside. Thromboxane B2 formation caused by Triwaglerin was suppressed by mepacrine, imipramine and indomethacin. R59022 and TMB-8 caused a synergistic inhibitory effect against Triwaglerin-induced aggregation. These data suggest that Triwaglerin activates platelets in a unique action which is independent of formation of thromboxane A2 and PAF, or release of ADP.     

Induction of lesions in deoxyribonucleic acid by low molecular weight substances from normal and colicin E2-treated cells of Escherichia coli.

A fraction containing a variety of low molecular weight substances was extracted into 80% aqueous acetone from both a colicin E2-treated cell culture of Escherichia coli and an untreated one. The extract was divided into five fractions by Sephadex G15 chromatography. One of them, Fraction B, was separated into three subfractions by Sephadex G10 chromatography. Two subfractions, Fraction BI and Fraction BII, were further fractionated by several chromatographic systems. DNA was incubated with an aliquot from each of these fractions and was then analyzed by sedimentation in an alkaline sucrose density gradient. The activity which caused a decrease in the sedimentation coefficient of the DNA was found in some of these fractions. The activity from colicin E2-treated cells was compared with that from untreated ones. It was revealed that colicin E2 induces some increases in the activity toward DNA in one of the subfractions, Fraction BI, and also causes the appearance of a new species in another fraction, Fraction BII, which potentiates the activity in Fraction BI. These colicin E2-induced changes appeared at 5 min after the addition of colicin E2. The possible significance of such reactions for the action of colicin E2 are discussed.     

Comparative studies on the nonprotein but nondialysable sugar-peptide fraction isolated from the plasma of various classes of vertebrates.

1. The nonprotein but nondialysable ninhydrin-positive substances isolated from plasma of some species belonging to four classes of vertebrates (fishes, amphibians, birds and mammals) were studied. 2. In all species studied (Cyprinus carpio, Salmo gairdneri, Rana esculenta, Rana temporaria, Gallus domestica and Rattus rattus) a similar group of nondialysable ninhydrin-positive substances may be isolated from the plasma by Sephadex G-25 filtration followed by dialysis. 3. Thin-layer Sephadex G-25 electrophoresis disclosed in birds (Gallus domestica) the lack of one ninhydrin-positive band contrary to the species of other classes of vertebrates. 4. Electrochromatography showed in all species studied an alteration in the composition of the basic peptide fractions and in birds the lack of one neutral peptide.     

Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae)

The high mortality caused by Crotalus durissus terrificus snake venom is mainly due to crotoxin, which acts on the neuromuscular junction inhibiting the mechanism mediating acetylcholine release, thus leading to motor and respiratory paralysis and subsequently to animal death. We recently demonstrated that the aqueous extract (AE) of Tabernaemontana catharinensis can inhibit the lethal activity of C. d. terrificus venom. Eight fractions, PI to PVIII, were obtained by gel filtration of the extract on Sephadex G-10, and assayed for lethality and cytotoxicity. Fraction PVII [2.0 mg/100 g rat/50 microl saline solution (ss)] injected intramuscularly (i.m.) 20 s after the venom (240 microg) or crotoxin (200 microg/50 microl ss) neutralized the lethal activity of 2 LD50 of both. Fractions PI, PVI and PVIII (5.0 mg/100 g rat/50 microl ss) presented potent antitumoral activity in vitro against cells from human breast carcinoma (SK-BR-3) after 24 h incubation, as measured by Mosmann colorimetric method. Fraction PVII contains 12-methoxy-4-methylvoachalotine as its major component. These results demonstrate that the antivenom and antitumoral activities of the AE of T. catharinensis are exerted by different substances present in fraction PVII and fractions PI, PVI and PVIII, respectively, whose characteristics are distinct in terms of staining and Rf when analyzed by thin layer chromatography. The results also show that a preliminary fractionation by Sephadex G-10 gel filtration is a good option as a first step for isolation of biologically active substances from T. catharinensis.     

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.     

Promotion of tea pollen tube growth by incubated solution of rapeseed cakes

The existence of growth promoters in rapeseed cakes (an organicfertilizer) was tested by measuring tube elongation in pollenculture in vitro. Growth of tea pollen tubes on an agar mediumcontaining sucrose and boron was strikingly promoted by addingthe solution obtained from incubated rapeseed cakes to the medium.Addition of a solution diluted to 1/300 stregnth to the agarmedium increased the pollen tube elongation to 153–170%of the control. Maximum activity was found with the solutionin which rapeseed cakes had been incubated at 30?C for 2 weeks.The promotive substances were not soluble in ethyl ether, ethylacetate or n-butanol, but were soluble in water, and diffusedout by dialysing against distilled water. These substances werefound to be soluble in 50% methanol, and were separated intothree fractions by Sephadex G-25 gel filtration. The promotivesubstances were different from several known growth regulators,amino acids and mineral nutrients. (Received August 31, 1979; )     

Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands.

Two proteins with gonadotropin activity have been isolated from a highly purified chum salmon (Oncorhynchus keta) gonadotropin preparation (G-75 Fraction II) by chromatography on DEAE Bio Gel A. These gonadotropins exhibited distinct behaviour in polyacrylamide gel electrophoresis, chromatography on Sephadex G-75 superfine, and ratios of cAMP stimulation in immature rainbow trout ovaries and testes. Rechromatography of G-75 Fraction II on Sephadex G-75 superfine gave a symmetrical protein peak with a coincident cAMP activity profile. Repeated freezing and thawing elicited a shift in the cAMP activity profile toward the trailing edge of the protein peak. Data are discussed in terms of two gonadotropin molecules which respond differently to phase changes. Charge polymorphism was exhibited by isoelectric focusing in polyacrylamide gels of one of the DEAE fractions. Five UV absorbing bands were observed which stimulated cAMP production in immature rainbow trout gonads. Three of these bands increased adenyl cyclase activity in trout ovaries and testes. One of the bands stimulated cAMP production primarily in trout testes and the other stimulated trout ovaries, providing evidence for two gonadotropins, each of which is sex specific.     

Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.