Three glycoproteins with antimutagenic activity identified in Lactobacillus plantarum KLAB21

Antimutagenic substances were purified from a culture supernatant of Lactobacillus plantarum KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity against N-methyl-N'-nitro-N-nitrosoguanidine on Salmonella enterica serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.     

Evaluation of antioxidant activities and antimutagenicity of turmeric oil: a byproduct from curcumin production

Curcumin removed turmeric oleoresin (CRTO) was extracted with hexane concentrated to get turmeric oil, and that was fractionated using silica gel column chromatography to obtain three fractions. These fractions were analyzed by GC and GC-MS. Turmeric oil contained aromatic turmerone (31.32%), turmerone (15.08%) and curlone (9.7%), whereas fractions III has aromatic turmerone (44.5%), curlone (19.22%) and turmerone (10.88%) as major compounds Also, oxygenated compounds (5,6,8-10) were enriched in fraction III. Turmeric oil and its fractions were tested for antioxidant activity using the beta-carotene-linoleate model system and the phosphomolybdenum method. The fraction III showed maximum antioxidant capacity. These fractions were also used to determine their protective effect against the mutagenicity of sodium azide by means of the Ames test. All the fractions and turmeric oil exhibited a markedly antimutagenicity but fraction III was the most effective. The antioxidant effects of turmeric oil and its fractions may provide an explanation for their antimutagenic action.     

The preparation of highly enriched fractions of binucleated rat hepatocytes by centrifugal elutriation and flow cytometry.

A procedure is described for the isolation of highly enriched fractions of binucleated hepatocytes from rat liver. Liver cells isolated by EGTA and collagenase perfusion were initially subjected to centrifugal elutriation and second to flow cytometry coupled with Hoechst 33342 staining. The elutriation step yielded hepatocyte fractions which contained almost entirely mononuclear diploid cells and fractions enriched in binucleate hepatocytes. The fractions with the highest proportion of binucleated hepatocytes contained between 50 and 56% of these cells. Subsequent flow cytometric cell sorting yielded fractions which contained greater than 80% binucleated cells. These cells were viable in culture as demonstrated by the immunohistochemical detection of bromodeoxyuridine incorporation.     

Decontamination ofFusarium mycotoxins,nivalenol, deoxynivalenol,and zearalenone,in barley by the polishing process

Korean dehusked and unhusked barley naturally contaminated withFusarium mycotoxins were polished using a Satake Grain Testing Mill. The pearled barley and bran fractions with different degrees of polishing were analyzed for nivalenol (NIV) and deoxynivalenol (DON) by gas chromatography with an electron capture detector, and for zearalenone (ZEN) by high-performance liquid chromatography with a fluorescence detector. NIV was detected in all the pearled barley fractions, but DON and ZEN were not detected in ≥27 % pearled barley fractions from dehusked barley and ≥36% pearled barley fractions from unhusked barley. However, for all degrees of polishing, NIV, DON, and ZEN were detected in bran fractions. The levels of NIV, DON, and ZEN in the bran fractions increased several fold over the original barley. Polishing was effective in removing DON and ZEN from the naturally contaminated barley, but not NIV.     

MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN RAT SYNAPTOSOME FRACTIONS DURING NEONATAL DEVELOPMENT

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.     

In vitro recognition of alloantigens: nature of responding and stimulating cells.

Immunosuppressive and immunostimulatory activity of human cancer ascitic fluids has been examined using the in vitro primary plaque-forming cell response (PFR) to sheep erythrocytes (Mishell-Dutton assay). We have prepared three fractions of ascitic fluid by precipitation with ammonium sulfate. Suppressive activity occurred in the fraction which was insoluble at 50% of saturation but not those fractions which precipitated at 30 or 80% of saturation. The fractions which were precipitated at 30 and 80% were stimulatory in the assay system. Normal human serum also had suppressive activity in the fraction precipitated at 50% of saturation but not as much as was found in ascitic fluid. Serum did not yield any fractions with stimulatory activity.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1 in Rat Nerve Myelin

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.     

The nature of the non-ultrafilterable glycosaminoglycans of normal human urine

1. The non-ultrafilterable acidic glycosaminoglycans from pooled urine of normal men, aged about 20, were isolated and characterized. The isolation procedure included digestion with sialidase and pronase, and fractionation by stepwise elution from an ECTEOLA-cellulose column. The glycosaminoglycans in each fraction were separated from each other by preparative electrophoresis in sodium barbital buffer and in barium acetate. 2. Approximate relative amounts of the different glycosaminoglycans were: chondroitin sulphate 60%, chondroitin 2%, hyaluronic acid 4%, dermatan sulphate 1%, heparan sulphate 15% and keratan sulphate 18%. Chondroitin sulphate-dermatan sulphate hybrids seemed to occur in trace amounts. 3. Chondroitin sulphate, heparan sulphate and keratan sulphate were heterogeneous with respect to degree of sulphation. Two distinct groups of chondroitin sulphate fractions were found, with sulphate/hexosamine molar ratios of about 0.5 and 1 respectively. The sulphate/hexosamine molar ratios in the heparan sulphate fractions varied from 0.5 to 0.9; the N-sulphate/hexosamine ratio was about 0.5 in all fractions. The sulphate/hexosamine molar ratios in the keratan sulphate fractions varied from 0.2 to 0.7.     

Distribution of phosphatidylinositol biosynthetic activities among cell fractions from rat liver.

The distribution of activities for synthesis of phosphatidylinositol among cell fractions from rat liver was determined. Activity was concentrated in endoplasmic reticulum; rough and smooth fractions were nearly equal. Golgi apparatus exhibited a biosynthetic rate 44% that of endoplasmic reticulum. Plasma membranes and mitochondrial fractions were only 6% as active as endoplasmic reticulum. Thus, endoplasmic reticulum and Golgi apparatus fractions from rat liver catalyze the net synthesis of phosphatidylinositol in vitro, whereas plasma membrane and mitochondrial fractions do not.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

东北次生杨桦林土壤碳氮动态特征

土壤分级组分是研究其碳氮动态的基础,次生杨桦林作为东北地区主要的天然林类型,目前相关数据的欠缺状态要求对此进行深入研究。为此,采集0—10cm、10—20cm、20—30cm长白山次生杨桦林土壤,通过土壤颗粒组分物理化学分级方法,将土壤分成5种组分:沙和稳定团聚体土壤组分(SA)、酸不溶土壤组分(AI)、易氧化土壤组分(EO)、颗粒态土壤组分(P)和可溶性土壤组分(S),进而分析了不同组分的质量分数、碳氮含量、碳氮分配比例及红外光谱5类官能团相对含量,旨在探讨次生杨桦林土壤固碳、氮供应机制。结果显示,接近90%的土壤质量集中在稳定组分AI(66.21%)和SA(22.11%)上,导致稳定组分中碳截获量最大(占土壤总碳量的2/3),而且其C/N比活跃组分(P和EO)大2—9倍;与碳不同,由于活跃组分中N含量比稳定组分大4—80倍,致使活跃组分P和EO氮的分配比例最大,分别占土壤总氮的33.1%和26.0%;除了占土壤质量很少的P和S外,组分间以及组分内的碳氮间多具有显著相关关系。这种土壤碳、氮在不同组分间贮存方式的差异使得土壤碳储存稳定性更高、而N肥力供应更快速。伴随不同组分碳氮储存的变化,不同组分间红外官能团存在显著差异,AI组分中绝大多数官能团相对含量均最低,而P和S组分中绝大多数官能团相对含量均较高,绝大多数官能团相对含量与碳含量、氮含量呈现显著的正相关关系,反映了官能团具有维持土壤碳氮的功能。同时,官能团与土壤C/N具有显著相关关系,反映出组分官能团相对含量的高低具有指示组分化学活性高低的作用。研究发现对于林分土壤的碳截获与氮供应的机制阐明具有重要的科学意义,这为深入了解东北次生杨桦林碳氮动态及对未来气候的响应提供基础数据。     

Subcellular Localization of Enzymes of Carbon and Nitrogen Metabolism in Nodules of Medicago sativa

Alfalfa (Medicago sativa L. cv. Vernal) nodules were separatedinto host plant fractions and fractions of rhizobial originby differential centrifugation and sedimentation equilibriumcentrifugation. Both NAD- and NADP-linked isocitrate dehydrogenase(70%, 90%) and glutamate dehydrogenase activities (90%, 83%)were located primarily (percent total nodule activity) in thefractions of plant origin and their specific activities werehighest in the fractions of plant origin. More than 50% of thenodules' total activity of both glutamine synthetase and NAD-glutamatesynthase and greater than 90% of the total glutamate oxaloacetatetransaminase activity was located in plant fractions. However,the fractions of rhizobial origin had the highest specific activitiesof glutamine synthetase and glutamate synthase. (Received September 5, 1981; Accepted December 7, 1981)     

The Lipid and Protein Staining Properties of Sudan II, III and IV and Their Components

Saturated solutions of commercial Sudan II, III and IV in 60% ethanol were found to stain heat-coagulated fat-free purified human serum albumin and defatted whole serum. The protein staining components were isolated and identified by means of paper chromatography on mineral oil-impregnated filter paper with 95% ethanol as the developing agent. The brownish and yellowish fractions which migrated rapidly in this system stained proteins well, whereas the red, pink and orange fractions, which migrated slowly, stained lipids only. The solubility and staining power of the slowly migrating lipid coloring fractions remained satisfactory in absence of the rapidly moving protein staining fractions.     

Antibacterial and antifungal activities of andrachne cordifolia muell

The crude methanolic extract of Andrachne cordifolia Muell. (Euphorbiaceae) and its various fractions in different solvent systems (chloroform, ethyl acetate and n-butanol) were screened for antibacterial and antifungal activities. Crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities against the tested pathogens. Highest antibacterial activity was displayed by both chloroform and ethyl acetate fractions (100%) followed by the crude extract (68%) against Salmonella typhi. Similarly, crude extract and its subsequent fractions showed mild to excellent activities in antifungal bioassay with maximum (76%) antifungal activity against Microsporum canis by the chloroform fraction followed by the crude extract (65%).     

Antibacterial and antifungal activities of teucrium royleanum (Labiatea)

The crude methanolic extract and subsequent fractions of Teucrium royleanum (Labiatea) were screened for antibacterial and antifungal activities. Against tested pathogens, crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities. Highest antibacterial activity was displayed by the ethyl acetate fraction against S. typhi (100%), against E.coli (76.7%) and against P. aerugenosa (70.8%) followed by the chloroform fraction against S. typhi (85.7%). Similarly, the crude extract and its subsequent fractions showed mild to excellent activities in the antifungal bioassay with maximum antifungal activity against M. canis (87%) by the chloroform fraction followed by the ethyl acetate (71%) and n-butanol (70%) fractions.     

Metabolism of triglyceride-rich nascent rat hepatic high density lipoproteins

Nascent high density lipoprotein (HDL) and nascent very low density lipoprotein (VLDL) were isolated from rat livers that had been perfused with [3H]glycerol to label the triglyceride. When injected into intact rats, the labeled HDL-triglyceride disappeared as rapidly as the VLDL-triglyceride, with only 10% of the injected label remaining in the plasma after 30 min. The protein moiety of nascent HDL was labeled with [35S]methionine in a similar fashion and the labeled nascent HDL was separated into nonretained (NR) and retained (R) fractions by heparin-Sepharose affinity chromatography. When injected into rats, 55% of the injected label in nascent fraction NR and 72% of that in nascent fraction R was recovered from plasma at 30 min, compared to only 10% of the triglyceride label from unfractionated nascent HDL, indicating dissociation of triglyceride and apolipoprotein clearance. The plasma decay curves for both triglyceride and protein were biexponential. By 5 min, 15% of the 35S label remaining in plasma represented apoE and apoC that had been transferred from nascent HDL fractions NR and R to the d less than 1.063 g/ml fraction of plasma. Plasma HDL was labeled in vivo with [35S]methionine, separated into fractions NR and R, and the clearance of the two plasma HDL fractions was compared with that of the corresponding nascent HDL fractions. Except for a faster rate of removal of the nascent HDL fractions during the first 5 min, the serum decay curves were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with (14)C-labelled carbohydrate or with (3)H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-(14)C]glucose or [G-(3)H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s(0) (25,w) values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B-C, which were chemically, biosynthetically and immunologically very similar.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.