In vivo and in vitro antitumoral effects of an extract from Vibrio cholerae in different murine models. I. Effect in vivo of bacterial extract from Vibrio cholerae on the development of different murine tumoral grafts]

A bacterial extract from Vibrio cholerae, so called DGZ, is shown to prevent transplantation of sarcoma 180 and Lewis lung carcinoma. A 4 days DGZ treatment (4 x 100 micrograms) triggers reject of the graft by 40% of the mice. The mice which have rejected a graft once after treatment, cannot be grafted lately or, at least, the tumor growth is delayed. The association with cisplatinum (4 x 25 micrograms) allows to protect 90 to 95% of the mice.     

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.     

Effects of hypothalamic extract and corticosterone on incorporation of 14C-phenylalanine into newly synthesized ACTH-like protein in vitro.

The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, 'cold' rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of 14C-phenylalaine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10(-4) and 10(-6) M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with 3/5, 1, and 2 HSME equivalents added.     

DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract.

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.     

尾叶远志抗氧化活性研究

采自贵州兴义的尾叶远志(Polygala caudata Rehd.et Wils.)根,经乙醇提取,再用不同溶剂萃取和柱层析分离,得到了12个不同的组分.用比色法研究了各组分还原三价铁离子及清除脂性自由基DPPH能力,采用化学发光法观察各组分清除羟自由基和超氧阴离子的活性.研究发现,尾叶远志各组分均有一定的清除自由基作用,并且其活性成分可能主要集中在PC-BuOH组分.     

Bone matrix studies. Influences of parathyroid extract, calcitonin, and cholecalciferol and of rickets and its treatment.

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by parathyroid extract. Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamin D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Biologically active constituents of a polyphenol extract from Geranium sanguineum L. with anti-influenza activity

From the aerial roots of the medicinal plant Geranium sanguineum L. a polyphenol-rich extract with strong anti-influenza activity has been isolated. To investigate its active fractions, the extract was partitioned by solvents with increasing polarity. The n-BuOH fraction contained the majority of the in vitro antiviral activity; the EtOAc fraction was the most effective one in vivo. A bioassay-directed fractionation of the n-BuOH and EtOAc fractions was performed to obtain information about the nature of the chemical components of the plant extract, responsible for the antiviral effect. The individual constituents were identified by spectroscopic methods and comparison with authentic samples and by HPLC. The cell-toxic and virus-inhibitory effects of the fractions and some individual polyphenol compounds, found in Geranium sanguineum L., were studied using the replication of representative influenza viruses in cell cultures. This study showed that the presence of a variety of biologically active compounds as well as the possible synergistic interactions between them seem to be decisive for the overall antiviral effect.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.     

Acaricidal activity of petroleum ether extract of neem (Azadirachta indica) oil and its four fractions separated by column chromatography against Sarcoptes scabiei var. cuniculi larvae in vitro

The petroleum ether extract of neem oil and its four fractions separated by column chromatography was diluted at different concentrations with liquid paraffin. The acaricidal bioassay was conducted using a dipping method. The results indicated that the median lethal concentration (LC50) of the petroleum ether extract (at the concentration of 500.0ml/l) was 70.9ml/l, 24h after treatment. At concentrations of 500.0, 250.0, 125.0, 62.5 and 31.2ml/l, the median lethal times (LT50) of the petroleum ether extract were 8.7, 8.8, 10.8, 11.5 and 13.1h, respectively. Thin-layer chromatography (TLC) showed that the petroleum ether extract of neem oil separated into four fractions (F1-F4). Acaricidal activity of 68.3% and 100.0% in the F2 and F4 was confirmed. These results suggest that petroleum ether extracts of neem oil and its four fractions possess useful acaricidal activity in vitro.     

Cytological and cytochemical effects of a liver extract from an adult rat on chick embryo hepatocytes and fibroblasts cultivated in vitro]

The effects of an extract from adult liver on chick embryo hepatocytes and fibroblasts cultivated in vitro are studied with cytological and quantitative cytochemical methods. This substance inhibits the multiplication of hepatocytes adn prevents these cells from entering into the S phase of the cycle. No effect is observed on the chick embryo fibroblasts. Not any morphological alterations are observed in the two cell types.     

An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro.

The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 micrograms of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 micrograms of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-beta may be present in this acid/ethanol extract of skeletal muscle.     

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G₀/G₁ phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.     

Comparative study of radical scavenger activities of crude extract and fractions from Cuphea carthagenensis leaves.

This study investigated the superoxide anion and hydroxyl radical scavenger properties, as well as the inhibition of lipid peroxidation by the crude hydroalcoholic extract (CE) and the butanolic (BF) and ethyl acetate (EAF) fractions of Cuphea carthagenensis leaves. In a enzymatic system of O2- production (xanthine/xanthine oxidase system) the CE, EAF and BF (0.1-100 microg ml(-1)) were effective at inhibiting both uric acid formation and NBT reduction by O2(-1). In the non-enzymatic system of O2- generation, the CE and fractions were effective only at the concentration of 100 microg ml(-1). The CE, EAF and BF were also evaluated for their ability to scavenge hydroxyl radicals and/or to chelate iron. The results showed that CE, BF and EAF from C. carthagenensis (0.1-100 microg ml(-1)) were able to inhibit deoxyribose degradation in a concentration-dependent manner. CE was more potent than the fractions. In a hydrophobic system, increasing concentrations of CE, EAF and BF (0.1-100 microg ml(-1)) caused graded inhibition of lipid peroxidation of rat liver homogenate. The EAF displayed the lowest median inhibitory concentration. The present study suggests that an extract (CE) and fractions (EAF and BF) from C. carthagenensis leaves are significant sources of phenolic compounds with antioxidant activity in vitro and may have important health effects, for example, in cardiovascular disease.     

Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds

In vitro antioxidant activities of three selected Indian red seaweeds - viz., Euchema kappaphycus, Gracilaria edulis and Acanthophora spicifera were evaluated. Total phenolic content and reducing power of crude methanol extract were determined. The antioxidant activities of total methanol extract and five different solvent fractions (viz., petroleum ether (PE), ethyl acetate (EA), dichloromethane (DCM), butanol (BuOH) and aqueous) were also evaluated. EA fraction of A. spicifera exhibited higher total antioxidant activity (32.01 mg ascorbic acid equivalent/g extract) among all the fractions. Higher phenolic content (16.26 mg gallic acid equivalent/g extract) was noticed in PE fraction of G. edulis. Reducing power of crude methanol extract increased with increasing concentration of the extract. Reducing power and hydroxyl radical scavenging activity of E. kappaphycus were higher compared to standard antioxidant (alpha-tocopherol). The total phenol content of all the seaweeds was significantly different (P<0.05). In vitro antioxidant activities of methanol extracts of all the three seaweeds exhibited dose dependency; and increased with increasing concentration of the extract.     

Selective effect of fractions of a yeast extract (PCO) on normal and malignant cells

The selective action of two fractions of PCO (a yeast extract) on normal and malignant cells was demonstrated. In vivo, the mitotic activity of malignant cells was inhibited by the methanol-insoluble fraction of PCO, whereas the methanol-soluble fraction caused no inhibition. The in vitro studies, however, showed inhibition of mitoses with both fractions. The malignant cells employed in vitro and in vivo were the Krebs-2 and Ehrlich carcinomas. The nonneoplastic cells tested in vitro were established cultures of epithelial-like cells from murine bone marrow and thymus, and corneal epithelium and peripheral blood in vivo.     

Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.