Liposomes as a carrier of genetic information

Incorporation of genetic material into the bilayer lipid vesicles (liposomes) and the subsequent transfer of liposomal content into cells or protoplasts appear to be a promising technique for transfer of genetic information. The following three methods are most frequently used to incorporate DNA into liposomes lipid microinjection into aqueous phase, multistep treatment of the lipid suspension by ultrasonication, Ca2+ ions and EDTA, reverse phase evaporation. Viral particles, chromosomes, nuclei, viral nucleic acids, plasmids and chromosomal DNA can be successfully transferred into animal and plant protoplasts by the described technique. Successful transformation of a number of microorganisms (Neurospora, E. coli, B. subtilis, Streptomyces, Mycoplasma) with the liposome incorporated DNA has also been reported. Transformation frequency can be considerably increased by optimizing the conditions of liposome formation or of liposome-protoplasts interaction.     

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.     

Light as an environmental filter in fen vegetation

Questions: 1. To what extent does light availability differ among fen plant communities? 2. To what extent does light coincide with productivity and moisture gradients? 3. Does light act as an important environmental filter in natural and transformed riparian landscapes? Location: Current data from the Biebrza Valley, NE Poland; literature data from the T?eboň area, Czech Republic and four sites in the western and southern Netherlands. Methods: Relative light intensity (RLI) was measured in vertical profiles, next to vegetation relevés accompanied by measurements of above‐ground biomass, summer groundwater level, N and P content in vegetation, pH and soil redox potential. Data derived from literature included profiles of RLI, biomass and vegetation records. Relationships between RLI and biomass and between species distribution, RLI and other variables were examined by regression analysis and CCA. Four traits were analysed: ability to spread clonally, seed weight, maximum height of adults and time of commencement of flowering. Results: RLI at ground level varied from < 1% in reed beds and tall herb meadows to > 60% in sedge‐moss communities and litter meadows. RLI was largely determined by the standing crop and explained a large part of variation in species occurrence. The combinations of analysed functional traits were constrained by the communities’ light profiles. Conclusion: Light availability is related more closely to site fertility than to hydrological regime. This confirms that hydrological regime and productivity should be analysed separately with regard to their effect on species distribution in wetlands. Limited light availability seems the major environmental control of the distribution of low growing and late flowering species.     

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.     

Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana

Background

Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.

Methodology and Principle Findings

Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

Conclusions and Significance

Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.     

Al-ATP as an intracellular carrier of Al(III) ion.

1. Using 27Al and 31P NMR spectroscopy in conjunction with an Al lactate aqueous reagent at pH 7.2, Al complexes of ATP and of phospholipids were characterized in synthetic-aqueous and organic-phospholipid chemical systems and in the intact human red blood cell. 2. The observed 31P NMR chemical shifts of the Al-ATP complex in aqueous laboratory preparations or the intact human red blood cell were, respectively, alpha phosphate, -11.53 delta; beta phosphate, -22.65 delta; and gamma phosphate, -10.95 delta. 3. The observed complexed 27Al chemical shift was -2.22 delta. 4. The relative affinities for Al of the phospholipids determined from 31P NMR spectroscopic titrations were PA much greater than Cl much greater than PS greater than PG approximately equal to PI greater than PE plus approximately equal to PE much greater than SPH greater than PC.     

Use of bottom ash of waste coal as an effective microbial carrier

An experiment was done to determine the efficacy of waste bottom ash as an effective microbial carrier. Bottom ash found to be a suitable microbial carrier. The average of viable cells of Paenibacillus polymyxa GS01 (as a test biocontrol agent) in bottom ash samples was about 10(8) cfu/10 ± 2 mg. The surface of bottom ash coated with 5% PVA w/v was most effective for improvement of cell viability. TSB medium containing 50 mg/L of MnSO(4) · H(2)O was the best for spore production of P. polymyxa GS01. Thus waste bottom ash coating with 5% PVA is likely to be suitable for use as a microbial carrier.     

An archaeal histone-like protein as an efficient DNA carrier in gene transfer

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.     

Spectral information as an orientation cue in dung beetles

During the day, a non-uniform distribution of long and short wavelength light generates a colour gradient across the sky. This gradient could be used as a compass cue, particularly by animals such as dung beetles that rely primarily on celestial cues for orientation. Here, we tested if dung beetles can use spectral cues for orientation by presenting them with monochromatic (green and UV) light spots in an indoor arena. Beetles kept their original bearing when presented with a single light cue, green or UV, or when presented with both light cues set 180° apart. When either the UV or the green light was turned off after the beetles had set their bearing in the presence of both cues, they were still able to maintain their original bearing to the remaining light. However, if the beetles were presented with two identical green light spots set 180° apart, their ability to maintain their original bearing was impaired. In summary, our data show that ball-rolling beetles could potentially use the celestial chromatic gradient as a reference for orientation.     

Light as an Energy Source in Continuous Cultures of Bacteriorhodopsin-Containing Halobacteria

The role of light as an energy source for slightly aereated cultures of halobacteria was studied, using continuous cultures with low nutrient concentrations and a low oxygen supply. A series of experiments were carried out with non-illuminated and differently illuminated cultures and with different oxygen transfer rates. Under low oxygen availability, light proved to be a decisively important energy source that allowed the populations to reach higher growth rates and much higher population densities. Oxygen influenced the growth over only a minimal level, below which neither the illuminated nor the dark cultures were affected by the oxygen transfer rate. From these results, it appears that the bacteriorhodopsin-mediated energy supply could have a very important role for the ecology of halobacteria in their microaerophilic habitats. In the illuminated cultures, cells that originated purple colonies on plates appeared. These cells, which could be bacteriorhodopsin-constitutive mutants, are now being studied.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

Acetylaspartate as an extramitochondrial physiological carrier of acetyl coA for fatty acid synthesis

Aminooxyacetate, a transaminase inhibitor, suppresses the enrichment in radioactivity found in the fatty acids of animals receiving 2, 4-¹⁴C citrate in comparison with 1, 5-¹⁴C citrate. On the other hand 3H from N-acetyl-³H aspartate is significantly incorporated into fatty acids $\text{in vivo}$ or in presence of liver supernatant fractions. Our results indicate that citrate seems to be an effective carrier of acetyl CoA for fatty acid synthesis mainly in the rat liver and that acetylaspartate may be an other physiological carrier of acetyl CoA outside the mitochondria.