Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

Treatment of leachate from a solid waste landfill site using a two-stage anaerobic filter

Raw leachate was treated using a two-stage upflow anaerobic filter process. Leachate from a solid waste landfill site, which received both municipal and industrial wastes, contained high organic matter (17-21 g/L COD, 13-14 g/L BOD, and 3.5-4.6 g/L volatile acids), and low metal (Zn and Fe) concentrations. Depending on sampling time, leachate composition and characteristics varied considerably. At an organic loading up to 4 g COD/day(2) media area, the BOD and COD removal percentages were 98 and 91%, respectively. The biofilters were also effective for metal removal. However, the filter effluent contained a high concentration of ammonia. System overloading was characterized by the accumulation of large quantities of volatile acids and by a now ratio of alkalinity/volatile acids, resulting in low COD removal and reduced gas production. Once the first filter was upset, the second stage could only partially respond to the volatile acids accumulated in the effluent of first filter.     

Application of hydroelastic waves of the Chinese traditional medicine solution to the traumatotherapy

We have used hydroelastic waves to treat the closed trauma of the soft tissue. The Shu Huo Jiu (S. H. J.) which is the Chinese traditional medicine alcohol, was used as the fluid medium for generating the pressure waves. The biomechanical model was established and analysed. Both animal and human tests have been made. A practical system was designed, constructed and clinically tested to treat the closed trauma, such as the bruise, contusion, sprain etc.. This system was found to be effective.     

De novo and maintenance DNA methylation by a mouse plasmacytoma cell DNA methyltransferase

A DNA methyltransferase of Mr = 140,000 that is active on both unmethylated and hemimethylated DNA substrates has been purified from the murine plasma-cytoma cell line MPC 11. The maximal rate of methylation was obtained with maintenance methylation of hemimethylated Micrococcus luteus or M13 DNAs. At low enzyme concentrations, the highest rate of de novo methylation occurred with single-stranded DNA or relatively short duplex DNA containing single-stranded regions. Strong substrate inhibition was observed with hemimethylated but not unmethylated DNA substrates. Fully methylated single-stranded M13 phage DNA inhibited neither the de novo nor the maintenance reactions, but unmethylated single-stranded M13 DNA strongly inhibited the maintenance reaction. The kinetics observed with hemimethylated and single-stranded substrates could be explained if the enzyme were to bind irreversibly to a DNA molecule and to aggregate if present in molar excess. Such aggregates would be required for activity upon hemimethylated but not single-stranded DNA. For de novo methylation of duplex DNA, single-stranded regions or large amounts of methyltransferase appear to be required. The relative substrate preference for the enzyme is hemimethylated DNA greater than fully or partially single-stranded DNA greater than fully duplex DNA.     

青岛文昌鱼卵卵黄粒内的一种亚微结构

在文昌鱼卵及胚胎发育过程中,卵黄粒内存在一种结构,形态上有7—9个亚单位排列呈环的管,类似“微管”,其直径为500—700(?),具“负染色”性(醋酸铀和柠檬酸铅染色)。推测其成分可能为糖-蛋白和脂蛋白及中性脂肪的复合体;其功能可能与建成细胞成膜物质有关。     

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.     

Characterization of the oligosaccharide alditols from ovarian cyst mucin glycoproteins of blood group A using high pressure liquid chromatography (HPLC) and high field 1H NMR spectroscopy

A combination of reverse phase and normal phase high pressure liquid chromatography has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of a blood group A ovarian cyst mucin glycoproteins. Fourteen compounds, ranging in size from a monosaccharide to a decasaccharide, have been isolated preparatively using a Zorbax C-18 reverse phase column eluted with water and a MicroPak AX-5 normal phase column eluted with aqueous acetonitrile. The purity of the products and their structures were determined from the fully assigned high field proton NMR spectra. The resonances of exchangeable amide protons, observed by the Redfield selective pulse sequence in H2O, were assigned by decoupling to the resonances of H2 of the 2-acetamido sugars. Nuclear Overhauser effects were used to establish the relationship of the anomeric protons and those of the aglycone. In exception to earlier proposals that nuclear Overhauser effect on irradiation of the anomeric proton should always be observed at the proton attached to the aglycone carbon, we find that for the linkage of GalNAcp(1----3)Gal, nuclear Overhauser effect on irradiation of the alpha-anomeric proton resonance is observed not at H3 but at H4 of galactose. A combination of NMR methods and enzymatic degradation was employed to determine the structures of 13 different oligosaccharides of which seven have not previously been reported. These oligosaccharides, which terminate with beta-Gal, alpha-Fuc, beta-GlcNAc, and alpha-GalNAc, account for 75% of the total glycoprotein carbohydrate, the remainder being isolated as a mixture of glycopeptides and a high molecular weight polysaccharide whose NMR spectrum implies a simple repeating subunit structure closely related to that of the oligosaccharides.     

Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2

The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme.