Fraction 3 Research Articles

Antidiabetic and antioxidant potency evaluation of different fractions obtained from Cucumis prophetarum fruit

Context: Cucumis prophetarum Linn. (Cucurbitaceae) fruit is used for inflammatory-related problems and is proved to be possessing anticancer and hepatoprotective effects.Objective: The present investigation was to study the effect of different fractions of C. prophetarum on antidiabetic and antioxidant activity.Materials and methods: Aqueous crude extract (CE) of C. prophetarum fruits was fractionated into water soluble fraction 1 (F1), chloroform fraction 2 (F2), basic fraction 3 (F3), and neutral fraction 4 (F4) by acid–base extraction. CE and its fractions at different doses (0.02–0.1 mg/mL) were subjected to antidiabetic (α-amylase and α-glucosidase inhibition assays) and antioxidant (DPPH, superoxide radical scavenging (SO) and metal chelation) evaluation.Results: F1 exhibited effective antidiabetic activity (p < 0.05) with an IC50 value of 20.6 and 59.9 µg/mL. The activity decreased in the order of CE > F4 >F3 > F2, according to α-amylase assay, which were the same, with the exception of the rank order of F4 and CE, as the α-glucosidase assay. Furthermore, F1 (IC50 = 73 µg/mL) showed better reducing ability than CE >F4 >F2 > F3 (IC50 = 78–272 µg/mL), according to the DPPH assay. In SO and metal chelation assays, F1 showed the highest activity (IC50 = 101 and 147 µg/mL), respectively; the activity decreased in the order of CE >F4 >F3 > F2 (IC50 = 126–469 µg/mL) for SO and 194–944 µg/mL for metal chelation assay.Conclusion: The results indicate that F1 possesses potent in vitro antidiabetic and antioxidant activities.

Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

Antibacterials and phytochemicals investigations of Chromolaenaodorata (L.F.) King and Robinson (asteraceae) from Sabah, Malaysia

Antimicrobial properties and phytochemical constituents in leaf extract of Chromolaenaodorata was evaluated in this study. C. odorataleaves were subjected to liquid-liquid extraction by using methanol, hexane, ethyl acetate, chloroform, buthanol and water. All extract partitions were tested for antibacterial activity against five Gram positive and Gram negative bacteria by using disc diffusion method. Crude methanolic extract (CME), ethyl acetate extract (EAE) and chloroform extract (CE) showed good antibacterial properties against the tested bacterial strains. However, only the CE was further separated using silica column chromatography. About 10 semi purified fractions was obtained and fraction 2 (F2) showed consistent inhibitory zones against all bacterial tested. Phytochemical investigations on the extract partitions and fractions showed the presence of alkaloids, flavonoids, tannins, polyphenols, saponins and triterpenoids. Fraction F2 was subjected to GC-MS analysis to characterised the bioactive compounds. The GC-MS spectral data has identified 10 major compounds which are hexachloro-ethane, n-nonylaldehyde, methyl-4-oxooctanoate, longiverbenone, 2-butenal,2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl), neophytadiene, phytol, dihydro-neoclovene, 2,6-ditert-butylquinone and aromadendrene

Distillation fraction-specific ecotoxicological evaluation of a paraffin-rich crude oil

Crude oil is a complex mixture of petroleum hydrocarbons (PHC) with distinct chemical, physical and toxicological properties relevant for contaminated site risk assessment. Ecotoxicological effects of crude oil distillation fractions on luminescent bacteria (Vibrio fischeri), earthworms (Dendrobaena hortensis) and invertebrates (Heterocypris incongruens) were tested using two spiked soils and their elutriates. Fraction 2 (F2) had an equivalent carbon number (ECN) range of >10 to 16, and F3 from >16 to 39. F2 showed a substantially higher ecotoxicological effect than F3 for Vibrio and Dendrobaena. In contrast, severe inhibition of Heterocypris by the poorly soluble F3 is attributed to mechanical organ blockage. Immediate sequestration of PHC to the organic matter-rich soil effected reduced toxicity for all organisms. This study indicates that a more differentiated consideration (i) of PHC mixtures based on ECN range and (ii) of model soil properties employed for ecotoxicity testing should be included into PHC-contaminated site risk assessment.

Isolation of α-glucosidase Inhibitors Produced by an Endophytic Fungus, Colletotrichum sp. TSC13 from Taxus sumatrana

Colletotrichum sp. have potential to act as antidiabetic agent, due to its alpha-glucosidase inhibitory. Therefore, the objective of present study was to isolate and identify the bioactive compounds responsible for the alpha-glucosidase inhibitory activity in Colletotrichum sp. TSC13. The methanol extract of TSC13 mycelia, was partitioned with n-hexane, chloroform and ethyl acetate. The n-hexane fraction exhibited the strongest alpha-glucosidase inhibitory activity. Column chromatography of this fraction resulted in 8 sub-fractions (F1-8). Fraction 3 (F3) which showed 71.4 +/- 2.4% inhibition was analysed further. Analysis using GC-MS after methylation of F3 and comparison to spectra databases and confirmation using authentic sample standards showed that F3 had two saturated fatty acid methyl esters, palmitic acid and stearic acid methyl esters and three unsaturated fatty acid methyl esters, oleic acid, linoleic acid and linoleinic acid methyl esters. Unsaturated fatty acids showed higher activity than the saturated fatty acids and the methyl esters form of unsaturated fatty acids showed slightly less active than the free acids. Further analysis using an ethyl acetate extract, it was confirmed that most of the fatty acids were present in the form of free acids. Therefore, it was concluded that the alpha-glucosidase inhibitor compounds in Colletotrichum sp. TSC13 were unsaturated fatty acids. This is the first report that a Colletotrichum sp. from T. sumatrana has alpha-glucosidase inhibitory activity.

Purification, partial characterization and anti-inflammatory characteristics of lotus (Nelumbo nucifera Gaertn) plumule polysaccharides

A novel lotus plumule polysaccharide (LPPS) was purified, characterised and cultured with RAW264.7 macrophages to evaluate its anti-inflammatory characteristics. LPPS was purified using Sepharose 6B gel filtration and dissolved into two major components, fraction-1 (F1) and fraction-2 (F2). The molecular weights of native F1 and F2 were approximately distributed at >2,000 and 25.7kDa, respectively. The total protein and carbohydrate constituent ratios in LPPS, F1, and F2 were 30.0±0.9% vs. 70.0±0.9%, 30.1±2.6% vs. 69.9±2.6%, and 96.5±6.1% vs. 3.5±6.1% (w/w), respectively, suggesting that F1 may be a major proteo-polysaccharide component and F2 a glycoprotein constituent in LPPS. Pro-/anti-inflammatory (IL-6/IL-10) cytokine secretion ratios by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly decreased by F1 and F2 treatments, particularly by F2, in a dose-dependent manner under a preventive experimental model. This study suggests that purified components, F1 and F2 from LPPS, have strong anti-inflammatory effects on LPS-induced inflamed macrophages in a preventive manner.

ANTI-FATIGUE AND ANTIOXIDATIVE ACTIVITIES OF PEPTIDES ISOLATED FROM MILK PROTEINS

Milk proteins were hydrolyzed with chymotrypsin and trypsin. Effects of hydrolysis conditions were studied through L9 (34) orthogonal experiment, and optimization hydrolysis conditions (substrate concentration, 25% (w/v); hydrolysis temperature, 45C; hydrolysis pH, 8.0; hydrolysis time, 2 h) were obtained. Six peptide fractions were separated from hydrolyzates by ultra-filtration and sephadex G-15 gel filtration chromatography. Anti-fatigue activity (swimming endurance tests) of each fraction was determined. Fraction 3 (F3) possessed longest swimming time. The anti-fatigue activity of the F3 peptide was evaluated. Loaded swimming time, serum blood urea nitrogen (SBUN), blood lactic acid and hepatic glycogen of mice were determined. F3 peptide can significantly increase the loaded swimming time and hepatic glycogen of mice, and decrease SBUN and blood lactic acid after swimming of mice. The antioxidant activity of the F3 peptide was also evaluated. F3 peptide had capacity to scavenge hydroxyl radical and superoxide anion radical. PRACTICAL APPLICATIONS Milk protein has been widely used as a material for peptides production by enzymatic hydrolysis. However, few anti-fatigue peptides from milk protein were reported. This work was found that the peptides hydrolyzed from milk protein had both anti-fatigue and antioxidant activities. It is to be expected that milk protein-derived anti-fatigue and antioxidant activity peptides would have no undesirable side-effects. As a consequence, the milk protein hydrolyzates with anti-fatigue and antioxidant activity could be applied in functional foods or beverages designed for persons suffering from diseases caused by fatigue, reactive oxygen species (ROS) and free radicals.

Paraoxonase 1 protects macrophages from atherogenicity of a specific triglyceride isolated from human carotid lesion

Human atherosclerotic lesions contain oxidized lipids that facilitate further oxidation of macrophages, LDLs, and oxidative stress (OS)-sensitive markers and inhibit the antiatherogenic enzyme paraoxonase 1 (PON1). Our aim was to isolate and identify the oxidizing agent in a human atherosclerotic lesion lipid extract (LLE) and to explore the mechanisms of oxidation and of PON1's effect on the oxidizing agent. Of the five main fractions separated from the LLE, only fraction 2 (F2) promoted macrophage reactive oxygen species (ROS) production via a mechanism requiring mitochondrial involvement, whereas the NADPH oxidase system was not involved. Incubation of F2 with PON1 abridged the former's peroxide value and reduced its capacity to oxidize OS markers. The active agent was a triglyceride composed of palmitic, oleic, and linoleic acids, with 0.3% of its linoleic moiety in oxidized form. Incubation of either F2 or an identical synthetic triglyceride with PON1 reduced their ability to oxidize macrophages, without affecting cellular accumulation of triglycerides. We conclude that macrophage ROS production by LLE occurs in the presence of a specific triglyceride and requires mitochondrial involvement. Lipid peroxide in the triglyceride can also facilitate lipid autoxidation. Both atherogenic pathways are suppressed by PON1, which acts as an antiatherogenic element.

Trypanocidal potentials of Azadirachta indica seeds against Trypanosoma evansi

The trypanocidal potentials of Azadirachta indica seeds methanolic extract (NSME) against Trypanosoma evansi was examined. In vitro studies with the NSME 100mg/ml, 50mg/ml and 25mg/ml immobilized the parasites within 3min, 8min and 14min respectively. In vivo experiments in infected rats at various dosage with NSME expressed transient ability of clearing the parasites in the infected blood. Thin layer chromatographic (TLC) separations of the NSME gave 4 fractions in toluene and ethyl acetate [1:0.25] solvent system on TLC of which only fraction 3 (F3) retained the trypanocidal properties which cleared the parasites in the infected rats for 14 days. The high performance liquid chromatography (HPLC) analysis of NSF F3 revealed the presence of Azadirachtins A and B as active components. The NSF F3 manifested prophylactic potency at a dose of 500mg/kg/day×3/7. The packed cell volume (PCV) of the group administered 500mg/kg/day×3/7 NSF F3 and normal control (NC) had no significant difference. The NSF F3 also inhibited Phospholipase A2 enzyme in a dose-dependent pattern.

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Heat-Shock Protein-70, -27 and Peroxiredoxin-2 Show Chaperone Function in Sickle Red Cells: Evidences from Transgenic Sickle Cell Mouse Model.

Sickle cell disease (SCD) is a worldwide-distributed hereditary red cell (RBCs) disorder characterized by the pathologic hemoglobin-S (HbS). The acute clinical manifestations of SCD are strictly related to HbS polymerization, to generation of dense RBCs, to their interaction with the abnormal activated vascular endothelial cells and to amplified inflammatory response. To identify new biomarkers associated with acute SCD events, we studied the RBCs membrane proteome from human SCD erythrocytes (n=20) fractioned according to density compared to normal RBCs (n=20), we divided RBCs into 2 fractions: fraction 1 (F1) corresponding to the least dense RBCs (density < 1.074) and fraction 2 (F2) corresponding to the densest RBCs (density > 1.095). None of the patients were under hydroxyurea treatment and they did not receive transfusion in the sixmonths before the study. Bi-dimensional electrophoresis (2-DE) followed by MS-analysis of RBCs F1 and F2 from both control and SCD subjects was carried out and 65 proteins differently expressed were identified. We focused on molecular chaperones. In SCD RBCs, we found that the amount of HSP27, HSP70 and Prx-2 recruited to the membrane was higher than in controls, Prx-2 was present as monomers and dimmers that were more abundant in SCD RBCs than in normal controls. Then, we exposed F1 and F2 fractions from normal and SCD patients to in vitro cellular stress (deoxygenation). In sickle RBCs, deoxygenation induced1.increase membrane recruitment of HSP27 in F1,2.modulation of HSP70 membrane binding in F2;3.reduction of Prx-2 monomers with increase Prx-2 dimerization in F1, but slightly changes in Prx-2 monomers in F2.These data suggest that in SCD RBCs HSP-27, -70 and Prx-2 are recruited to the membrane in response to cellular stress, acting as molecular chaperones to assist proteins in regaining their functional conformation. In order to evaluate their role in vivo and because increase serum levels of HSP70 has been recently reported in few SCD patients, we used transgenic sickle cell SAD mice as a model for SCD and exposed to hypoxia (8%-O2) respectively for 2–12–48 hours, mimicking acute human SCD vaso-occlusive-crisis (VOCs). We studies SAD mice and wild-type mice (C57/B6, WT) divided into groups of 6 animals each. We evaluated HSP70, HSP27 and Prx-2 expression on RBCs membrane, hematological parameters, RBCs density, and cation content. We found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 hours and 48 hours (hrs) of hypoxia, while Prx-2 membrane binding was modulated during hypoxia. At the different time schedule, we observed a marked reduction in red cell K+ at 12 and 48 hrs hypoxia, associated with significant increase of the dense RBCs at 48 hrs hypoxia. Our data indicate that in SCD, HSP70, HSP27 and Prx-2 membrane recruitment is modulated in vivo during acute VOCs, supporting their novel role as RBCs membrane protein protectors and as new markers of severity of RBCs membrane damage during acute sickle VOCs.

Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and Antigenic Protein Fractions

This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240min at 50°C. The highest hydrolysis of WPC was observed after 240min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (β-lactoglobulin) in WPC were degraded within 60min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of β-lactoglobulin, α-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2mg/kg), followed by hydrolysates derived with Flavourzyme (0.14mg/kg), trypsin (0.14mg/kg), and papain (0.08mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4·7H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC.

Mutation of Cys105 Inhibits Dimerization of p12CDK2-AP1 and Its Growth Suppressor Effect

p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.

Purification and partial characterization of phospholipases A2 from Bothrops asper (barba amarilla) snake venom from Chiriguaná (Cesar, Colombia)

Components with phospholipase A2 activity were isolated by gel filtration and cationic exchange chromatography from the venom of Bothrops asper snakes from Chiriguana, Colombia (9°22´N; 73°37´W). Five fractions were obtained by the gel filtration, and PLA2 activity was found in fraction 3 (F3). In the cationic exchange chromatography, F3 showed eight components with PLA2 activity. Six of these components appeared as one band in polyacrylamide gel electrophoresis (SDS-PAGE). Fractions II and VII exhibited an optimal activity at pH 9 and 52oC. The optimum calcium concentration for fraction II was 48 mM and for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten constant (KM) was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes did not show any toxic activity (DL50) at 5.3 and 4 µg/g. The two fractions showed different kinetic constant (KM), calcium requirement, and substrate specificity for haemolytic activity.

Instrumental and bioanalytical measures of persistent organochlorines in blue mussel (Mytilus edulis) from Korean coastal waters.

Blue mussels (Mytilus edulis) collected from 34 locations along the south and east coast of Korea were analyzed for polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides. Maximum concentrations of PCBs and total OC pesticides were 98.5 and 20.5 ng/g, wet weight, respectively. Extracts were fractionated by Florisil chromatography and each fraction was screened for dioxin-like activity in vitro, using recombinant rat hepatoma cells (H4IIE-luc). Fraction 2 (F-2), which contained hexachlorocyclohexanes, chlordanes, p,p'-DDD, and p,p'-DDT, generally elicited significant dioxin-like activity compared to control, whereas Fraction 1 (F-1), which contained PCBs, p,p'-DDE, and hexachlorobenzene, did not. The greatest magnitude of dioxin-like response observed was 44% of the maximum response elicited by a 2,000 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard. The relatively low magnitudes of dioxin-like response observed for F-1 samples were consistent with the relatively low PCB concentrations. At concentrations equal to the maximum observed in the mussel samples, neither individual OC pesticides nor a mixture of OC pesticides yielded a significant dioxin-like response in the H4IIE-luc assay. Thus, the concentrations of OC pesticides in F-2 did not appear to have accounted for the dioxin-like activity observed. This suggests the presence of unidentified and/or unknown, acid-stable, dioxin-like compounds in F-2. This study suggests that in vitro bioassays are useful in assessing the contamination of mussels collected from coastal marine locations.

Application of The Gelatin Particle Indirect Agglutination Test in The Serodiagnosis of Human Opisthorchiosis.

Bithynia funiculata snail antigens were partially purified by Sephacryl S-200 gel filtration chromatography. Four fractions of B. funiculata snail antigens were obtained-fraction 1 (F1), fraction 2 (F2), fraction 3 (F3) and fraction 4 (F4), which Fl was chosen for the study since this is the most abundant and most reactive fraction. Levels of antibodies in sera of 85 patients with opisthorchiosis, 15 normal healthy individuals and 72 patients with other helminthic infections were assayed against crude antigens from O.viverrini adult worms and the Fl fraction using the gelatin particle indirect agglutination test (GPAT).For both antigens, the mean reciprocal titer in the sera of opisthorchiosis patients was significantly higher than sera from patients with other helminthic infections and normal healthy individuals (p <0.0001). In an attempt to search for another antigen in place of 0. viverrini antigens for the serodiagnosis of opisthorchiosis, the sensitivity and specificity of the two antigens were compared. It was shown that differences in the sensitivity and specificity were not evident. This study indicates that antigens from 0. viverrini adult worms were the most suitable antigens for serodiagnosis of opisthorchiosis by GPAT despite being crude antigens. However, antigens from partially purified B. funiculata snails (Fl) should prove to be useful for serodiagnosis of this disease since large amounts of antigens can be obtained with relative ease.

Studies on the venom components of the long-glanded coral snake, Maticora bivirgata

The venom of the Asian long-glanded coral snake, Maticora bivirgata, was fractionated into five fractions, S1–S5, by passing through a Sephadex G-50 column. Fraction S2 contains two phospholipases A 2, PLA 2 I and PLA 2 II, fraction S3 contains four cytotoxin homologues, maticotoxins A, C, D1 and D2, and fractions S4 and S5 contain a large amount (about 1 mg/specimen) of adenosine accompanied with smaller amounts of inosine and guanosine. The amino-terminal amino acid sequences of PLA 2 I, PLA 2 II and maticotoxin A suggest that Maticora bivirgata is closely related to Bungarinae, especially to genera Hemachatus and Naja.

Immunological responses to protein, carbohydrate and lipid fractions of Nocardia asteroides in mice.

The protein, polysaccharide and phospholipid constituents of Nocardia asteroides have been partially purified and their immunogenicity studied in mice. Humoral and cellular immune responses were demonstrated against a crude cytoplasmic protein fraction (CPF). Two fractions of CPF were prepared on a Sephadex G-200 column; a high mol. wt fraction, fraction-1(F1) was capable of eliciting both types of immune responses, whereas fraction-2(F2) behaved more like a hapten. Phosphatides elicited only humoral responses whereas polysaccharides were non-immunogenic.

Biochemical and allergenic properties of the house dust mite extract, Dermatophagoides pteronyssinus.

A further study has been made on the house dust mite extract, Dermatophagoides pteronyssinus, with emphasis on gel-filtrated fraction 2 (F2). The crude mite extract showed at least nine discs on polyacrylamide disc electrophoresis and contained less than 0.25% sialic acid and less than 0.5 mM hexosamine and no detectable uronic acid. From gel filtration of the crude extract a No 2 fraction (F2) with allergenic activity showed at least five components on SDS disc electrophoresis covering a molecular weight range of between 15,000 and 70,000. The major allergenic activity of F2 dissolved in pH 7-8 and 4-5 on an isoelectric focusing column. Affinity chromatography and lectins showed that allergenic activity did not relate to structures of N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. Allergenic activity of the crude extract was no affected by peptic digestion and the mite digest prepared by trypsin and pronase showed a similar fractionation and activity profile as crude extract.

Dissociation of chorioadenoma destruens from hydatidiform mole by urinary steroid analysis

Dissociation between hydatidiform mole (HM) and chorioadenoma destruens (CA) was attempted by gas chromatographic analysis of urinary steroids. In the chromatogram, fraction 2 (F2) including four pregnane steroids (17-hydroxypregnanolone, pregnanolone, pregnanediol, and pregnanetriol) reflected the biologic activity of molar human chorionic gonadotropin (hCG), and the ratio of F2 to fraction 1 (F1) (17-ketosteroids) was found useful in monitoring secondary growth of trophoblastic tumor: Urinary log F2/F1 after curettage declined quickly and unidirectionally in HM, but the same parameter exhibited a temporal rise in CA at 11 to 20 days after curettage because of the molar hCG from residual tumor tissue. The resolution between HM and CA at that stage (11 to 20 curettage days) was of the order of diagnostic use and superior to that with immunoreacive hCG. The data obtained are discussed in the light of pathophysiology of CA.