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.     

Localization of a sheep pineal antigonadotropin

An antigonadotropic substance was partially purified from aqueous extracts of sheep pineal bodies by gel filtration on Sephadex G-25 and ultrafiltration using the Amicon Diaflo membranes UM-2 and UM-05. When injected into male and female Charles River CD-1 albino mice the low molecular weight fractions from Sephadex G-25, and after ultrafiltration the UM-05 residue of these fractions, demonstrated significant effects on the reproductive system. These effects included the inhibition of compensatory ovarian hypertrophy in unilaterally ovariectomized adult mice, the reduction of ventral prostate weights in adult mice, and the reduction of gonad and accessory reproductive organ weights in young male and female mice.Further purification of the antigonadotropic substance was accomplished by gel filtration of the UM-05 residue on Sephadex G-10. The elution volume of the biologically active fraction did not correspond to that of synthetic melatonin applied subsequently to the same column. The antigonadotropic activity in the UM-05 residue was localized by paper electrophoresis and paper chromatography.     

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.     

Isolation and characterization of agglutinin receptor sites: II. Isolation and partial purification of a surface membrane receptor for wheat germ agglutinin

Four different chemical extraction procedures for the isolation of wheat germ agglutinin receptor sites from L1210 cells are described. Fractionation of the biologically active material on Sephadex G-200 columns in pyridine results in two major peaks, the lower molecular weight fraction having a higher inhibitory activity. Electrophoresis in polyacrylamide sodium dodecyl sulfate gels yields four bands. The most active fraction from Sephadex G-200 has an approximate molecular weight between 40000–60000. A preliminary analysis of the active material indicates the presence of sialic acid, neutral sugars and amino sugars, including N-acetylglucosamine.     

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.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.     

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.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

CONTROL OF GRANULOCYTE PRODUCTION: SEPARATION AND CHEMICAL IDENTIFICATION OF A SPECIFIC INHIBITOR (CHALONE)

Abstract. It has previously been shown by others that blood serum contains inhibitors of blood cell production acting on the proliferation of granulocy tic and erythrocytic precursor cells in the bone marrow. It is now shown that the active extract from calf blood serum can be further subfractionated into six different components, all of them exhibiting inhibitory effects on the proliferation of rat bone marrow cells in vitro. Ascitic fluid from rats treated intraperitoneally with polyvinylpyrrolidone contains inhibitors which apparently are the same as those found in calf serum.
It was further possible to demonstrate that only one of these inhibitors is contained in mature granulocytes where it is actively synthesized from amino acids and subsequently released into the surrounding medium. By chromatography on Sephadex G-25 of this conditioned medium the inhibiting substance could be obtained in relatively pure form being contaminated only by low amounts of two ninyhdrin-positive substances. the experiments allow the granulocytic inhibitor to be identified as a polypeptide with a molecular weight below 5000. the results suggest that this substance is the granulocytic chalone.     

LH inhibitory properties of aqueous extracts of rat pineal glands

Crude aqueous extracts of rat pineal glands markedly inhibited the 24 and 48 hour postcastration rise of serum LH in mature male rats. After partial purification by exclusion chromatography on Sephadex G-25 the LH inhibitory activity was found to reside in a volume not coinciding with that of melatonin, indicating that some substance other than this indole may be responsible for the anti-LH property in aqueous extracts of rat pineal glands.     

Pollen Tube Growth Inhibitors Involved in the Ovary of Self-Incompatible Japanese Pear

The growth inhibitors of pollen tubes in the pistils of Japanesepear ‘Chojuro’ were studied in vitro to elucidatethe physiological mechanism of self-incompatibility. Addition of water extracts from ovaries into a well dug in anagar medium containing sucrose and boric acid inhibited thegrowth of incompatible pollen tubes more strongly than thatof compatible ones. The substance, tentatively called Sinhibitor(self-inhibitor), was detectable in a relatively wide rangeof concentrations of the extract, from 10- to 60-fold dilution.However, it was absent in the stylar extract. S-inhibitor was stable, even though the extract was heated to100?C for 10 min. The chromatogram of the S-inhibitor followinga Sephadex G-10 gel filtration showed 2 peaks of inhibition:one peak corresponded to the peaks of protein and phenol (phenolsmay be conjugated with proteins), and the other to that of reducingsugar. The water extract from mature ovaries when diluted 10-fold wasa stronger growth inhibitor of incompatible pollen tubes comparedwith that from immature ones. This substance, tentatively calledA-inhibitor (adult-inhibitor), appeared to be different fromS-inhibitor. (Received May 20, 1986; Accepted December 22, 1986)     

Effect of different leukocyte pyrogen fractions on hemopoiesis

Fractionation of leukocyte pyrogen on a column of Sephadex G-75 made it possible to obtain separately the fraction stimulating the hemopoiesis and the fraction possessing the pyrogenic activity and inhibiting the hemopoiesis. Judging by the elution profile of Sephadex column G-75, substances of high molecular weight produced a stimulating action, and of low molecular weight--pyrogenic and inhibitory action. Possibly pyrogenic and inhibitory activities are connected with different substances. The nature of the inhibitory factor requires further investigation. It may be supposed that it is a substance of chalone type.     

Multiple forms of rat liver mitochondrial DNA polymerase.

Mitochondrial DNA polymerase (DNA polymerase mt) exists in two active forms. DNA polymerase present in crude extract (M-I) and ammonium sulfate precipitate (M-II) stages of purification sediments at 12.1S. The enzyme at the M-II stage of purification has a molecular weight of approximately 250,000 as determined by Sephadex G-200 chromatography in buffers of low ionic strength. In buffers containing 0.15 m NaCl, the enzyme sediments at 9.4S and has a molecular weight of approximately 190,000. When the enzyme is further purified on diethylaminoethyl cellulose (M-III stage of purification), the 9.4S activity predominates. Addition of a polymerase-free fraction from the M-III stage of purification changes the sedimentation coefficient of the enzyme from 9.4 to 12.1S.     

Isolation of Inhibitory Factor in Raw Milk Whey Active Against Propionibacteria

Preparative isolation of the active component(s) in skim milk whey inhibitory for propionibacteria was made by using (NH(4))(2)SO(4) salt fractionation. The crude preparation was further purified by Sephadex G-100 column separation. Disc-gel electrophoresis of the active peak from the Sephadex elution pattern (peak I) showed that this fraction contained almost all of the immune globulin in the column sample. The biologically inactive peaks did not contain any immune globulin. Starch-gel electrophoresis of the active peak revealed the presence of three separate immune globulin fractions. A correlation was also observed between hemolytic reaction of propionibacterial strains and relative resistance to whey inhibition. The investigation showed that one of the immune globulins of milk, pseudoglobulin, was mainly responsible for the suppressive activity of whey.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.     

PRESENCE OF 1-METHYLADENINE IN SEA URCHIN GONAD AND ITS RELATION TO OOCYTE MATURATION

A water extract of sea urchin ovary was found to induce maturation of starfish oocytes in vitro . The presence of the active substance was demonstrated in ovaries of the sea urchins, Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus , and the sand dollars, Clypeaster japonicus and Peronella japonica . The active substance was also contained in the testes of these echinoids. That the content of this substance increases during the reproductive season was demonstrated with Anthocidaris gonads. The active substance present in ovary or testis of the sea urchins was successfully extracted with 85% ethanol and purified with gel-filtrations on Sephadex G-15 columns after washing with chloroform and ether. The purified active substances were the same and were identified as 1-1-methyladenine by thin layer chromatography. 1-Methyladenine was found to be effective in inducing oocyte maturation in Anthocidaris crassispina in vitro . Therefore, 1-methyladenine seems to play an important role in oocyte maturation in echinoids as well as in asteroids.     

Isolation and partial chemical characterization of THF, a thymus hormone involved in immune maturation of lymphoid cells.

A procedure for isolation and characterization of the active principle of calf thymus is described. It consists of homogenization, ultracentrifugation and dialysis of the material. The active dialyzate is further purified by gel filtration on Sephadex G-10 and G-25 columns followed by anion exchange chromatography. The level of purification is assessed by isoelectric focusing on polyacrylamide gels. The active principle is a polypeptide of MW 3220. Determination of the amino acid composition revealed the presence of high proportion of acidic residues.     

Respiratory behaviour of sea-urchin spermatozoa. II. Sperm-activating substance obtained from jelly coat of sea-urchin eggs.

A sperm-activating substance (SAS) was obtained from the jelly coat of sea-urchin ova and its chemical properties were investigated in three sea-urchin species. The SAS was partially purified from the jelly coat of Pseudocentrotus eggs through several steps of purification by procedures consisting of charcoal adsorption, ion-exchange chromatography on DEAE-Sephadex A-25 column, and gel-filtration on Sephadex G-15 columns. The partially purified SAS was found to contain a ninhydrin-positive material and is inactivated by pronase digestion. The molecular weight of SAS was estimated as about 630 by gel-filtration through Sephadex G-25 and the isoelectric-point of SAS is located at about pH 5.3 by isoelectrofocusing method. The SAS is non-volatile, alcohol-soluble, and labile in a diluted alkaline or acid solution. The origin of SAS is discussed.