Photoenhancement of transfection efficiency using novel cationic lipids having a photocleavable spacer

New cationic lipids having an o-nitrobenzyl moiety as a photocleavable spacer between its hydrophilic and hydrophobic region were synthesized. To improve the efficiency of transfection with lipoplexes, after transfecting the cationic lipid aggregate/DNA complex, photoirradiation was performed. Photochemical decomposition of lipids would not only make the vector's membrane unstable to facilitate the fusion with endocytic vesicles, but also promote dissociation of cationic lipid-DNA complex, thus aiding the escape of DNA from the endocytic vesicles. Using a luciferase gene as a model, we show that UV irradiation of photoresponsive lipoplex-treated COS-1 cells induces a substantial increase in the efficiency of transfection. Herein, we show a novel photoresponsive gene delivery system.     

Influence of spacer length on heparin coupling efficiency and fibrinogen adsorption of modified titanium surfaces

Background  

Chemical bonding of the drug onto surfaces by means of spacer molecules is accompanied with a reduction of the biological activity of the drug due to a constricted mobility since normally only short spacer molecule like aminopropyltrimethoxysilane (APMS) are used for drug coupling. This work aimed to study covalent attachment of heparin to titanium(oxide) surfaces by varying the length of the silane coupling agent, which should affect the biological potency of the drug due to a higher mobility with longer spacer chains.     

Hormonal influence on uterine lipids.

The effects of oestrogen (OE), progesterone (P) and oestrogen plus progesterone (OEPP) on uterine lipids of adult ovariectomized rats were studied. Administration of gonodal hormones brought about considerable alterations in total lipids, mainly due to an increase in phospholipids and glycerides in the case of OE and P groups. Even though the total cholesterol remained unaltered, a marked alteration in its fractions was evident in all the groups. Oestrogen seems to decrease all the glyceride classes while progesterone induces accumulation ot triglycerides with concomitant decrease in mono- and diglycerides. Individual classes of phospholipids show marked alterations in their distribution in all the groups studied.     

Hormonal influence on testicular lipids.

The effects of progesterone and progesterone plus oestrogen (PPO) on testicular lipids of adult rats were studied. Treatment with progesterone over 7 days did not alter significantly the total lipids, cholesterol, glycerides and phospholipids. However, PPO administration brought about a significant elevation in total lipids, mainly contributed by the increase in triglycerides. The phospholipids and total cholesterol were not markedly affected by PPO treatment, but the individual classes of phospholipids showed marked alterations in their pattern of distribution. The esterified and free cholesterol fractions were found to be significantly altered by both the treatments. Progesterone appears to favour ester cholesterol accumulation by depleting the available free cholesterol. Oestrogen seems to increase the glycerides and change the concentration of phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine when administered with progesterone.     

Synthesis and transfection properties of novel non-toxic monocationic lipids. Variation of lipid anchor, spacer and head group structure

This report describes the synthesis and the transfection properties of novel monocationic non-toxic lipids. We have carried out structural variations in all three units of the transfection lipid, the lipid anchor, the spacer moiety and the positively charged head group. Our results lead to the conclusion that systematic modification of structural subunits is a promising way to enhance the transfection efficiency.     

Hydrolysis of oligoribonucleotides: influence of sequence and length.

The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis.     

Studies on the coupling rates in liquid-phase peptide synthesis using competition experiments.

Competition experiments were carried out in order to determine relative reaction rates, Vrel, for the peptide-forming step. Using the liquid-phase method of peptide synthesis a model system was developed to investigate the influence of coupling method, amino component, derivatization and solvent upon Vrel. According to a standard procedure, Vrel for all 20 commonly occurring amino acids were established. A strong dependence of Vrel upon steric effects of the coupling components was found. The data obtained may serve as a guideline for optimizing the reaction conditions in peptide synthesis.     

Studies on mixed monolayers of phospholipids and fusogenic lipids.

1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.     

Bivalent inhibitors of glutathione S-transferase: the effect of spacer length on isozyme selectivity

Glutathione S-transferases (GSTs) are cytosolic enzymes that catalyze the conjugation of glutathione with a variety of exogenous and endogenous electrophiles. High affinity, isozyme-specific inhibitors of GST are required for use as pharmacological tools as well as potential therapeutics. The design of selective inhibitors is hindered due to the broad substrate binding capabilities of the GST enzymes. GSTs are dimeric enzymes, and therefore offer a unique discriminator for achieving inhibitor selectivity: the distance between binding sites on each monomer unit as a function of its quaternary organization. Bivalent analogs of the non-selective GST inhibitor ethacrynic acid were prepared, and selectivity for the GST A1-1 isozyme over GST P1-1 (IC50 values of 13.7 vs 1022 nM, respectively) was achieved through the optimization of the spacer length between the ethacrynic acid ligand domains.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Sister chromatid exchange and the evolution of rDNA spacer length

The structures of rDNA spacers from several species have been characterized and virtually all have internally repeated sequences. Different numbers of these internal repeats are responsible for most spacer length variation. Because unequal recombination between these internal repeats will cause new length variation, while unequal exchange between rDNA copies will homogenize the variants, we modeled the interaction of these two processes. Two models were used to simulate both types of unequal exchange at the sister chromatid level. Both models indicate that a narrow range of relative recombination frequencies is required to produce levels of variability comparable to those published. One model puts a lower limit on the number of internal repeats, and the other puts both a lower and upper limit on the number of repeats. The model with both maximum and minimum constraints produces a distribution closer to actual spacer distributions. These results imply that small changes in recombination rates can generate the differences in numbers of length variants observed in different species.     

Phenoloxidase catalyzed coupling of catechols. Identification of novel coupling products.

Phenoloxidases from insect cuticle as well as from other sources oxidize catechols resulting in the formation of various coupling products. The two dominating products from 4-methylcatechol and the main product from N-acetyldopamine were purified and identified by means of plasma desorption and electron impact mass spectrometry and by 1H- and 13C-NMR spectroscopy. The main product from both catechols has a quinoid trihydroxybiphenyl structure, indicating oxidative coupling between a catechol and the corresponding trihydroxy derivative. The second product from 4-methylcatechol is a biphenyltetrol derivative, indicating oxidative coupling between two catechols.     

Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex

Many cell types in animals and plants are polarized, which means that the cell is subdivided into functionally and structurally distinct compartments. Epithelial cells, for example, possess an apical side facing a lumen or the outside environment and a basolateral side facing adjacent epithelial cells and the basement membrane. Neurons possess distinct axonal and dendritic compartments with specific functions in sending and receiving signals. Migrating cells form a leading edge that actively engages in pathfinding and cell-substrate attachment, and a trailing edge where such attachments are abandoned. In all of these cases, both the plasma membrane and the cytocortex directly underneath the plasma membrane show differences in their molecular composition and structural organization. In this chapter we will focus on a specific type of membrane lipids, the phosphoinositides, because in polarized cells they show a polarized distribution in the plasma membrane. They furthermore influence the molecular organization of the cytocortex by recruiting specific protein binding partners which are involved in the regulation of the cytoskeleton and in signal transduction cascades that control polarity, growth and cell migration.     

Synthesis and characterization of novel thiol-reactive poly(ethylene glycol) cross-linkers for extracellular-matrix-mimetic biomaterials

Synthetic extracellular matrix hydrogels can be used for three-dimensional cell culture, wound repair, and tissue engineering. Using the bifunctional electrophile poly(ethylene glycol) diacrylate (PEGDA), thiol-modified glycosaminoglycans and polypeptides can be cross-linked into biocompatible materials in the presence of cells or tissues. However, the rate of in situ cross-linking with PEGDA under physiological conditions may occur too slowly for clinical applications requiring a fast-curing preparation. To explore a wider range of cross-linking time courses, five homo-bifunctional PEG derivatives were synthesized and examined as cross-linking agents for thiol-modified derivatives of hyaluronan (HA). Thiol reaction rate constants were measured over a pH range of 7.4 to 8.6. The order of reactivity for the functional groups used was determined to be maleimide > iodoacetate > bromoacetate > iodoacetamide > acrylate > bromoacetamide, with rates increasing exponentially with increasing pH. The range of gelation times at physiological pH varied from less than 1 min to over 2 h. Addition of the cross-linkers to cell culture medium showed minimal cytotoxicity toward primary human dermal fibroblasts at concentrations anticipated during in situ cross-linking. Moreover, hydrogels prepared from thiol-modified gelatin and thiol-modified HA were biocompatible and supported attachment and proliferation of fibroblasts and hepatocytes.     

Extraordinary ribosomal spacer length heterogeneity in a neotyphodium endophyte hybrid: implications for concerted evolution.

An extraordinary level of length heterogeneity was found in the ribosomal DNA (rDNA) of an asexual hybrid Neotyphodium grass endophyte, isolate Lp1. This hybrid Neotyphodium endophyte is an interspecific hybrid between two grass endophytes, Neotyphodium lolii, and a sexual form, Epichlöe typhina, and the length heterogeneity was not found in either of these progenitor species. The length heterogeneity in the hybrid is localized to the intergenic spacer (IGS) and is the result of copy-number variation of a tandemly repeated subrepeat class within the IGS, the 111-/119-bp subrepeats. Copy number variation of this subrepeat class appears to be a consequence of mitotic unequal crossing over that occurs between these subrepeats. This implies that unequal crossing over plays a role in the concerted evolution of the whole rDNA. Changes in the pattern of IGS length variants occurred in just two rounds of single-spore purification. Analysis of the IGS length heterogeneity revealed features that are unexpected in a simple model of unequal crossing over. Potential refinements of the molecular details of unequal crossing over are presented, and we also discuss evidence for a combination of homogenization mechanisms that drive the concerted evolution of the Lp1 rDNA.     

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.     

Extreme length and length variation in the first ribosomal internal transcribed spacer of ladybird beetles (Coleoptera: Coccinellidae)

DNA sequences of the first ribosomal internal transcribed spacer (ITS1) were isolated from 10 ladybird beetle species (Coleoptera: Coccinellidae) representing four subfamilies (Coccinellinae, Chilocorinae, Scymninae, and Coccidulinae). The spacers ranged in length from 791 to 2,572 bp, thereby including one of the longest ITS1s and exhibiting one of the most extreme cases of ITS1 size variation in eukaryotes recorded to date. The causes of length variation were therefore analyzed. Almost no putatively homologous sequence similarities were identified for the taxa included. The only exception was for the subfamily Coccinellinae, which yielded sequence similarities in six regions of approximately 550 nucleotide positions, primarily at the 5' and 3' ends of ITS1. The majority of differences in ITS1 length between taxa could be attributed to the presence of repetitive elements with comparatively long repeat units. Repetition arose several times independently and was confined to the middle of the spacer which, in contrast to the 5' and 3' ends, had not been inferred in previous studies to be subject to functional constraints. These elements were characterized by high rates of evolutionary change, most likely as a result of high substitution rates in combination with inefficient homogenization across repeats. The repeated origin and subsequent divergence of "long" repetitive elements should thus be assumed to be an important factor in the evolution of coccinellid ITS1.