Isolation and frontier molecular orbital investigation of bioactive quinone-methide triterpenoids from the bark of Salacia petenensis.

The crude dichloromethane bark extract of Salacia petenensis (Hippocrateaceae) from Monteverde, Costa Rica, shows antibacterial and cytotoxic activity. Bioactivity-directed separation led to the isolation of tingenone and netzahualcoyonol as the biologically active materials. Also isolated from the extract were 3-methoxyfriedel-2-en-1-one (a new natural product) and 29-hydroxyfriedelan-3-one. The structures of these compounds were elucidated on the basis of NMR spectral analysis. Molecular orbital calculations have been carried out using the semi-empirical PM3 and Hartee-Fock 3-21G ab initio techniques on the quinone-methide nortriterpenoids tingenone and netzahualcoyonol, as well as on the nucleotide bases adenine, guanine, cytosine, and thymine. The molecular orbital calculations suggest that a possible mode of cytotoxic action of quinone-methide triterpenoids involves quasi-intercalative interaction of the compounds with DNA followed by nucleophilic addition of the DNA base to carbon-6 of the triterpenoid.

Synthesis and cytotoxic activity of a series of diacetylenic compounds related to falcarindiol.

The synthesis of a series of diacetylenic compounds related to the natural product falcarindiol has been carried out. Unsymmetrical diacetylenes were prepared by a modification of the Cadiot-Chodkiewicz coupling reaction, while a Glaser coupling was used to prepare symmetrical diacetylenes. These compounds have been tested for in vitro cytotoxic activity against Hep-G2, and H-4-II-E cell lines. Diacetylenes with additional unsaturation at C-1, 2, appended with hydroxyl groups at C-3 and C-8, or with long hydrophobic chains, exhibited IC50 values in the micromolar range.

A phytochemical investigation of Alchornea latifolia.

A phytochemical investigation of the chloroform leaf extract of Alchornea latifolia has been undertaken. Along with the triterpenoids taraxerone, friedelin, epifriedelinol, and taraxerol, the plant also contains seco-3,4-friedelin (dihydroputranjivic acid) (1) and seco-3,4-taraxerone (2). These A-ring-opened triterpenoids show in vitro cytotoxic activity against Hep-G2 and A-431 human cancer cell lines and are potent inhibitors of topoisomerase II.

Biological activity of the essential oil of Myrcianthes sp. nov. "black fruit" from Monteverde, Costa Rica.

The leaf essential oil of an undescribed species of Myrcianthes has been obtained from Monteverde, Costa Rica. The essential oil exhibits in vitro cytotoxic activity against Hep-G2 and SK-Mel-28 human tumor cell lines. A GC/MS analysis shows the essential oil to be composed of 2-heptanol, alpha-pinene, beta-pinene, limonene, cineole, and alpha-terpineol, alpha-Pinene, beta-pinene, and limonene account for the cytotoxic activity of the leaf essential oil of Myrcianthes sp. nov. "black fruit".

Lupeol is the cytotoxic principle in the leaf extract of Dendropanax cf. querceti.

The crude ethanol extract from the leaves of Dendropanax cf. querceti (Araliaceae) from Monteverde, Costa Rica, exhibits cytotoxic activity against Hep-G2, A-431, and H-4IIE tumor cell lines. The active component has been isolated by activity-directed separation and identified by 1H- and 13C-NMR spectroscopy as the triterpene lupeol. The mechanism of cytotoxic activity of lupeol has been determined to be inhibition of topoisomerase II.

A cytotoxic diacetylene from Dendropanax arboreus.

The crude ethanol extract from the leaves of Dendropanax arboreus (Araliaceae) from Monteverde, Costa Rica, exhibits cytotoxic activity against Hep-G2, A-431, H-4IIE, and L-1210 tumor cell lines, but is not toxic against normal hepatocytes. The active component has been isolated by activity-directed separation and identified by 1H- and 13C-NMR spectroscopy as the acetylenic compound cis-1,9,16-heptadecatriene-4,6-diyne-3,8-diol.

An antibacterial vitamin E derivative from Tovomitopsis psychotriifolia.

The crude ethanol extract from the leaves of Tovomitopsis psychotriifolia (Clusiaceae) exhibits antibacterial activity against Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa. The biologically active agent in the extract has been isolated by chromatographic techniques and identified by NMR spectroscopy as trans-delta-tocotrienoloic acid.

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model.

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

Protein metabolism in chicken muscle cell cultures treated with cimaterol.

Primary muscle cell cultures were prepared from the leg muscle of 12-d broiler chicken embryos. The partitioning agent cimaterol (10(-6) to 10(-10) M) was added on d 1 and each day thereafter, and cells were studied after 7 d in culture. Cimaterol had no effect at any level either on the percentage of nuclei within multinucleated myotubes or on the total number of nuclei within myotubes. At 10(-7) M cimaterol, the quantity of the myofibrillar protein fraction was increased by 25.1 +/- 8.0% (P less than .05) and the quantity of myosin heavy chain was increased by 30.9 +/- 4.5% (P less than .05). To understand the basis for the increase in myofibrillar protein, the incorporation rate of [3H]Leu was measured in pulse labeling experiments. The apparent synthesis rate of the soluble protein fraction and the crude myofibrillar fraction was not significantly increased by cimaterol; however, cimaterol levels greater than 10(-8) M caused a 10 to 12% increase (P less than .05) in the incorporation rate of [3H]Leu into myosin heavy chain. The effect of cimaterol on release of [3H]Leu from prelabeled protein also was assessed in pulse-chase experiments; the apparent rate of protein degradation was inhibited by 10 to 15% (P less than .05) at the higher levels of cimaterol. Dot blot analysis indicated that the quantity of myosin heavy chain mRNA was elevated in cimaterol-treated cultures. Thus, the increased quantity of myofibrillar proteins in embryonic broiler muscle cell cultures is the combined result of a stimulation in the rate of protein synthesis and an inhibition in the rate of protein degradation.

Genomic clones encoding chicken myosin heavy-chain genes.

A chicken genomic library was screened with a cDNA probe containing the 3' coding and noncoding portions of quail fast-twitch skeletal muscle myosin heavy chain (MHC). This probe hybridized to seven to nine bands on Southern blots of chicken genomic DNA, and 17 clones that hybridized to this probe were obtained from the genomic library. Partial restriction maps were constructed and probable orientation of transcription was determined for each of the 17 clones. These maps indicate the presence of at least 14 unique MHC genes or pseudogenes. Dot-blot hybridization analysis using DNA complementary to RNA from a variety of chicken tissues demonstrated that these genes are all related to the gene for sarcomeric MHC, and permitted tentative assignment of the tissue of expression for several of the MHC isoforms. To substantiate further the dot-blot data, a subclone of one of the genes (4b1), which showed significant homology with adult breast muscle RNA but which also showed weaker hybridization to RNA from other tissues, was sequenced. The sequence data verified that the clone contains a portion of a MHC gene, that it contains both 3' coding and noncoding regions, and that its predicted amino acid sequence is identical (with 96% nucleotide homology) to that of the 75-bp quail fast MHC cDNA clone published by Hastings and Emerson (1982). Thus, clone 4b1 contains a portion of one of the genes that is expressed in adult chicken breast skeletal muscle tissue.

Screening of a bovine genomic library for myosin heavy-chain genes.

Restriction enzyme digests of bovine genomic DNA were hybridized against a .37-kilobase (kb) quail embryonic myosin heavy-chain (MHC) copy deoxyribonucleic acid (cDNA) probe; containing both translated and nontranslated regions surrounding the 3' end of the gene. These experiments revealed seven to eight different bands of hybridization, indicative of multiple genes of MHC in the bovine genome. Additionally, a bovine genomic recombinant DNA library was screened with the .37-kb probe. Of the 10(6) phage screened, 11 clones containing portions of the MHC genome were identified, and four were selected for further analyses. Characterization of these four clones was carried out by constructing partial restriction enzyme maps of the inserts using six restriction enzymes singly or in combination. Orientation of the inserts with respect to the arms of the vector and with respect to direction of transcription was determined by hybridizing the DNA fragments against either the .37-kb Pst 1 fragment of pcC128 or to the .23-kb Pst I fragment of pcC128. The .23-kb fragment is located upstream from the .37-kg fragment and contains only coding sequence. Therefore, the differential hybridization pattern of these two probes provided a means for determining the probable direction of transcription. These data provide evidence for a myosin multigene family in cattle, as well as illustrating that the organization of these genes around the 3' end is unique for each of the genes analyzed.

Structural analysis of myosin genes using recombinant DNA techniques.

Myosin, the major protein of the myofibril, consists of two heavy chains with a molecular weight (MW) of 200,000, complexed with four light chains of MW 17,000 to 21,000. Both the heavy and light chains exhibit polymorphisms that are tissue-specific and developmental stage-specific. Myosin heavy chains and light chains appear to be represented in the genome as multigene families in various species, including chickens, cattle, humans, rats, rabbits and nematodes. Myosin heavy-chain proteins have a high amino acid sequence homology among isoforms, and the genes for each isoform likewise exhibit a high degree of nucleotide sequence conservation. The myosin heavy-chain genes have a complex structure and contain up to 65% intervening sequence composed of up to 20 or more individual introns. Partial sequence data, transcriptional orientation and tissue of expression have been determined for several myosin heavy-chain genes. The use of recombinant DNA and associated techniques will eventually yield definitive information on the control of expression of each individual gene, as well as factors that regulate expression of closely related isoforms.

Identification of human hepatoma-defined cell surface molecules.

BALB/c mouse splenocytes from mice immunized with cells of the human hepatoma line Hep G2 were fused with SP2/0-Ag 14 mouse myeloma cells. Two monoclonal antibodies recognizing antigenic determinants (Hag-1, Hag-2) of hepatoma cell surface molecules were investigated. Analysis of immunoprecipitates by sodium dodecyl sulfate (SDS) gel electrophoresis revealed that the Hag-1 antigenic determinant is born ona 115, kD MW glycoprotein, and that the second antibody immunoprecipitates a group of surface proteins with MW of 230 kD, 79 kD, 23 kD, and 20 kD from human hepatoma cells. These antigenic determinants are present on cell lines derived from other human tumors, thus neither of the antibodies is hepatoma-specific; cross-reactivity with human colorectal carcinoma and some mammary carcinoma cell lines is notable. Using indirect immunofluorescence on frozen sections Hag-1 was detected in one of three liver biopsies tested whereas Hag-2 was demonstrated in all three. Both antigens were detected in sections of human kidney with Hag-2 localized to the proximal tubules.

Monoclonal antibody to human epidermal growth factor.

A monoclonal antibody directed to a species-specific determinant of human epidermal growth factor (h-EGF) was obtained by fusing murine myeloma cells with BALB/c mouse splenocytes sensitized to h-EGF. This antibody, referred to as 863.D4, did not react with either rat or mouse epidermal growth factor or with 11 other polypeptide hormones tested as shown by solid-phase radioimmunoassay (SPRIA), and immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scatchard analysis of the antibody binding to purified h-EGF revealed an apparent equilibrium dissociation constant of 1 X 10(-8) M. The antibody blocked both the binding of h-EGF and h-EGF stimulation of 3H-thymidine incorporation into DNA by greater than 90% in confluent cultures of human foreskin fibroblasts.

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2- to 6-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. 12-O-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF. It did not inhibit the binding of 125I-labeled EGF to these cells. The stimulation of enzyme activity by EGF was not inhibited by cycloheximide or actinomycin D, although these agents did cause a significant decrease in enzyme levels when added without EGF. Also, colchicine, chloroquine, ammonium chloride, and methylamine, compounds that inhibit EGF degradation in various cells types, did not interfere with the ability of EGF to elevate enzyme levels in the human hepatoma cells.