Nanoparticles of compacted DNA transfect postmitotic cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Do mothers-in-law matter? Family dynamics and fertility decision-making in urban squatter settlements of Karachi, Pakistan

The perspectives of mothers-in-law about intra-household decision-making, family size and family planning are investigated, and their views compared with those of their sons and daughters-in-law. Women (717 daughters-in-law), their husbands (717 sons) and their 522 mothers-in-law were interviewed in eight squatter settlements in Karachi, Pakistan. Decisions about the schooling and health care of children, and the purchase of jewellery, are perceived to lie within the nuclear family domain (i.e. husband and wife). There was a difference in mothers-in-law's, daughters-in-law's and sons' desire to have more children. Twenty-eight per cent of mothers-in-law versus 58%, of daughters-in-law did not want more grandsons/sons and 36%, of mothers-in-law versus 66% of daughters-in-law did not want more granddaughters/daughters. The difference was markedly greater among the mother-in-law/daughter-in-law pairs than in the mother/son pairs. Overall, the mother-in-law's role seems to be somewhat overshadowed by that of her son (family male member), except for limiting family size. It is suggested that mothers-in-law should be included in Information-Education-Communication (IEC) campaigns about family planning.     

Regioselective sulfonylation of 6,1',6'-tri-O-tritylsucrose through dibutylstannylation: synthesis of 4'-O-sulfonyl derivatives of sucrose

3-O-Mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (3) was synthesized via stannylation of 6,1',6'-tri-O-tritylsucrose with dibutyltin oxide in benzene, followed by treatment of the crude product with methanesulfonyl chloride in the presence of triethylamine in dichloromethane at 0 degrees C. A similar treatment of the tri-tritylsucrose in toluene, instead of benzene, yielded 4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (4) as the major product. The X-ray crystal structure of the corresponding acetyl derivative, 3-O-acetyl-4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-2,3,4-tri-O-acetyl-6-O-trityl-alpha-D-glucopyranoside (5), confirms the position and stereochemistry of the methanesulfonyl group at C-4 of the fructofuranosyl ring.     

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

Arbuscular Mycorrhizal Fungi Enhance Zinc and Nickel Uptake from Contaminated Soil by Soybean and Lentil

Generally, soils in Pakistan are deficient in P and N. Due to intensive cropping and irrigation, Pakistani soils have also become deficient in micronutrients such as Zn, Fe, Cu, and Mn. Arbuscular mycorrhizal fungi, which form symbiotic associations with roots of most land plants, are known to enhance uptake of P and trace elements such as Cu, Ni, Pb, and Zn. The present study was conducted to investigate the role of arbuscular mycorrhizae (AM) in uptake of nickel (Ni) and zinc (Zn) by crops viz. soybean (Glycine max (L.) Merrill) and lentil (Lens culinaris Medic). Zn and Ni were applied as ZnSO₄ 7H₂O and NiCl₂ respectively, in four concentrations (0.0, 1.0, 3.0, and 5.0 g kg^-1 soil). AM inoculum consisted of sand containing sporocarps, spores, and AMF infected root pieces from a pot culture of Glomus mosseae. Control plants received pot culture filtrate containing soil microflora minus AM fungal propagules. A significant difference (p < 0.05) was observed in the dry weights of roots and shoots of the mycorrhizal (M) and nonmycorrhizal (NM) cereal plants. The sievate-amended treatments did not stimulate plant growth to the same extent as the AM fungal amended treatments. Trace metals inhibited the extent of mycorrhizal colonization of the cereal roots. The concentrations of the trace metals in the plant tissues of 12-week old cereal plants were found significantly (p < 0.05) higher in M than NM plants. These results indicate that mycorrhize can be used as effective tools to supply sufficient Zn in generally Zn-deficient Pakistani soils and to ameliorate the toxicity of trace metals in polluted soils. The contents of Ni in mycorrhizal soybean plant tissues were higher than those in the mycorrhizal lentil plant tissues. The implications of these results in mycorrhizo remediation of agricultural soils are discussed.     

A High Through-put Procedure for Capturing Microsatellites from Complex Plant Genomes

A method is outlined for large-scale isolation and characterization of microsatellite sequences from complex plant genomes. The method presented here differs from the previously published procedures in the use of randomly sheared (nebulized) genomic DNA for adapter-ligation, rigorous removal of biotinylated oligos, and high-density colony blots for constructing enriched libraries. Using this method we have constructed cotton microsatellite enriched libraries with over 20% (high stringency screening) or 75% (by random sequencing). Thus far we have identified and sequenced over 500 cotton microsatellites using this procedure. The procedure can be used to generate enriched SSR libraries from genomic DNA in about one week. High throughput screening and automated DNA sequencing can be accomplished in less than one month.