Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (相似文献   

Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.      

NMR Studies of the Interaction of Bleomycin with (dC-dG)3

Abstract

The interaction of bleomycin A₂ and Zn(II)-bleomycin A₂ with the oligonucleotide (dC-dG)₃ has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the ¹H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected.     

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.     

Impact of ivermectin on onchocerciasis transmission: assessing the empirical evidence that repeated ivermectin mass treatments may lead to elimination/eradication in West-Africa

BACKGROUND: The Onchocerciasis Control Program (OCP) in West Africa has been closed down at the end of 2002. All subsequent control will be transferred to the participating countries and will almost entirely be based on periodic mass treatment with ivermectin. This makes the question whether elimination of infection or eradication of onchocerciasis can be achieved using this strategy of critical importance. This study was undertaken to explore this issue. METHODS: An empirical approach was adopted in which a comprehensive analysis was undertaken of available data on the impact of more than a decade of ivermectin treatment on onchocerciasis infection and transmission. Relevant entomological and epidemiological data from 14 river basins in the OCP and one basin in Cameroon were reviewed. Areas were distinguished by frequency of treatment (6-monthly or annually), endemicity level and additional control measures such as vector control. Assessment of results were in terms of epidemiological and entomological parameters, and as a measure of inputs, therapeutic and geographical coverage rates were used. RESULTS: In all of the river basins studied, ivermectin treatment sharply reduced prevalence and intensity of infection. Significant transmission, however, is still ongoing in some basins after 10-12 years of ivermectin treatment. In other basins, transmission may have been interrupted, but this needs to be confirmed by in-depth evaluations. In one mesoendemic basin, where 20 rounds of four-monthly treatment reduced prevalence of infection to levels as low as 2-3%, there was significant recrudescence of infection within a few years after interruption of treatment. CONCLUSIONS: Ivermectin treatment has been very successful in eliminating onchocerciasis as a public health problem. However, the results presented in this paper make it almost certain that repeated ivermectin mass treatment will not lead to the elimination of transmission of onchocerciasis from West Africa. Data on 6-monthly treatments are not sufficient to draw definitive conclusions.     

Near-infrared optical imaging of B16 melanoma cells via low-density lipoprotein-mediated uptake and delivery of high emission dipole strength tris[(porphinato)zinc(II)] fluorophores

Meso-to-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn(3)) near-infrared (NIR) fluorophores (lambda(em)(max) approximately 800 nm) can be rendered sufficiently amphiphilic to enable their facile incorporation into the hydrophobic core of the apo form of low-density lipoprotein (apo-LDL). These NIR fluorophores are notable in that they manifest low energy excited states polarized exclusively along the long axis of the supermolecule, broad spectral coverage of the visible and high energy NIR spectral domains, intense S(0)-->S(1) transition moments, and comparably large S(1)-->S(0) emission dipole strengths. The reconstituted LDL(PZn(3)) proteins can be used to deliver rapidly hundreds of copies of PZn(3) to a given murine B16 melanoma cell via LDL receptor-mediated endocytosis. PZn(3)-based NIRFs and their corresponding LDL(PZn(3)) proteins have been shown to display minimal cytotoxicity. Confocal NIR fluorescence microscopy evinces that B16 cells can be imaged at very low doses (approximately nM) of NIRF. The highly attractive photophysical properties of PZn(3) and closely related chromophores, coupled with their lack of toxicity and compatibility with uptake into apo-LDL and subsequent rapid delivery to B16 cells via LDLr-mediated endocytosis, suggest the potential utility of this platform for NIR optical imaging of cancer cells in vivo.     

Increases in NMR-visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells

DU145 human prostatic carcinoma cells were treated with the differentiating agents phenylacetate (PA) and phenylbutyrate (PB) and examined in perfused cultures by diffusion-weighted 1H and 31P nuclear magnetic resonance spectroscopy (NMR). PA and PB (10 mM) induced significant (>3-fold) time-dependent increases in the level of NMR-visible lipids and total choline in 1H spectra, and glycerophosphocholine levels in the 31P spectra, with the increases being greater for PB. These effects were accompanied by significant increases in cytoplasmic lipid droplets and intracellular lipid volume fraction as observed by morphometric analysis of Oil Red O-stained cells. PB treatment caused cell cycle arrest in the G1 phase and induction of apoptosis. In contrast, PA-treated DU145 cells showed an accumulation of cells in G2/M and no evidence of apoptosis. These results demonstrate that significant differences exist in the mechanism of PA and PB activity, although both compounds cause similar, but graded alterations in lipid metabolism. The simultaneous accumulation of mobile lipid and glycerophosphocholine suggests that PB and PA induce phospholipid catabolism via a phospholipase-mediated pathway. The mobile lipid accumulation following the induction of either apoptosis and cytostasis by related differentiating agents indicate that the presence of NMR-visible lipids may not be a specific event causally resulting from the induction of apoptosis.