S. H. Moss

Polycation-DNA complexes for gene delivery : a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

ACTION SPECTRA FOR INACTIVATION OF NORMAL and XERODERMA PIGMENTOSUM HUMAN SKIN FIBROBLASTS BY ULTRAVIOLET RADIATIONS

—Action spectra for UV‐induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254‐365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254‐313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.

Synthesis and biological evaluation of α-MSH analogues substituted with alanine

Abstract The influence of single amino acid replacements by alanine on the binding affinity and biological activity of α-MSH in B16 murine melanoma cells has been studied systematically. α-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of α-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4–9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met 4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe 7 , Arg 8 , and Trp 9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

Lipid peroxidation and other membrane damage produced in Escherichia coli K1060 by near-UV radiation and deuterium oxide.

Abstract— With accumulating evidence that the membrane is an important site for near‐UV inactivating events, we have investigated the effects of near‐UV radiation on the unsaturated fatty acid auxotroph E. coli K1060 following incorporation into the membrane phospholipids of fatty acids with varying degrees of unsaturation. Sensitivity, lipid peroxidation and membrane damage, as determined by 86Rb+ leakage, have been found to increase with increasing unsaturation in log‐phase cells. Similar experiments carried out in D2O also show an increase in sensitivity, lipid peroxidation and membrane damage, indicating that singlet oxygen may be responsible for such near‐UV‐radiation‐induced damage

THE SENSITISING EFFECT OF A SUNSCREENING AGENT, p-AMINOBENZOIC ACID, ON NEAR UV INDUCED DAMAGE IN A REPAIR DEFICIENT STRAIN OF ESCHERICHIA COLI†

Abstract. The use of repair deficient strains of bacteria for detecting mutagenic properties of chemical species is now an established technique. In this paper we present inactivation results obtained with Escherichia coli K12 AB2480 (uvr A rec A) which indicate that p‐aminobenzoic acid, a common component of sunscreening formulations, may increase the frequency of lethal damage induced in DNA when cell populations are exposed to near ultraviolet radiation.

Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium.

AbstractPurpose. The use of rapidly dividing in vitro cell culture systems to assess the efficiency of gene delivery is now recognised as a poor indicator of in vivo success. We investigated whether differentiated Caco-2 cell filter-cultures would make a more suitable model for studying gene transfer to an epithelium. Methods. Caco-2 cells were cultured on semi-permeable membrane filters into differentiated polarised monolayers. Monolayer differentiation was assessed by monitoring the transport of taurocholic acid. Cells at different stages of differentiation were transfected with DNA/DOTAP lipoplexes and later analysed for reporter gene activity. The uptake of radiolabled DNA was also evaluated at various stages of differentiation. Results. Caco-2 cultures developed a resistance to lipoplex-mediated transfection as early as three days, when some cells were still dividing and undifferentiated. As cultures matured, expression of reporter gene progressively decreased partly due to reduced internalisation of DNA. The resistance to transfection could be overcome in part by pre-treatment of monolayers with calcium chelating agents or surfactants. However, transgene expression in treated monolayers was still significantly lower than that in dividing cultures. Conclusions. Differentiated Caco-2 cells are a more appropriate model for gene-transfer studies to the intestinal epithelium because they demonstrate a resistance to transfection similar to that observed in vivo.

The melanocortin (MC3) receptor from rat hypothalamus: Photoaffinity labelling and binding of alanine-substituted α-MSH analogues

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of α‐MSH, [125I‐Tyr2,Nle4,d‐Phe7,ATB‐Lys11]α‐MSH.SDS‐PAGE followed by autoradiography showed a single band at 53–56 kDA for the native receptor of 35 kDA after deglycosylated with PNGase F, consistent with the predicted cDNA. Receptor binding studies with α‐MSH, γ‐MSH and [Nle4,d‐Phe7]α‐MSH established that α‐MSH and γ‐MSH had similar affinities while [Nle4,d‐Phe7]α‐MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved ‘core sequence’ (‐Met‐Glu/Gly‐His‐Phe‐Arg‐Trp‐) of MSH/ACTH peptides. The binding affinities of alanine‐substituted analogues of α‐MSH were determined to investigate the role of individual residues in ligand—receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K‐12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl‐3H]thymidine leakage and 86Rb+ leakage) after broad‐band (Black‐Light Blue) near‐UV radiation but not after far‐UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild‐heat (52°C) treatment of E. coli K‐12. An action spectrum for the release of 86Rb+ from E. coli K‐12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F37 values) obtained for this strain, shows that leakage of 86Rb+ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of 86Rb+ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near‐UV radiation can induce a damaging effect on the cells permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far‐UV radiation where DNA damage is known to be the main cause of lethality.

Influence of α-MSH terminal amino acids on binding affinity and biological activity in melanoma cells

The influence of the terminal amino acids of alpha-MSH on its biological action in B16 murine melanoma cells has been systematically studied. Fragments of alpha-MSH lacking various sequences of terminal residues were synthesized by solid-phase peptide synthesis and their binding affinity to melanoma cells was measured using a radioreceptor assay. Biological activity was determined by measuring both tyrosinase activity and melanogenesis. The relative affinities and activities of the fragments generally followed the same pattern as found previously in other assay systems (frog and lizard bioassay and Cloudman S91 mouse melanoma), with the three amino acids at each terminal not being essential for binding and biological activity, although the C-terminal amino acids 11-13 are more important than those in the N-terminus. The differences in biological activity between the fragments can be explained by their relative binding affinities for the receptor.

AN ACTION SPECTRUM FOR ULTRAVIOLET RADIATION‐INDUCED MEMBRANE DAMAGE IN Escherichia coli K‐12

A wild‐type Escherichia coli K‐12 strain was irradiated using monochromatic radiation in the range 254 to 405 nm. A measure of the cell membrane damage induced at each wavelength was investigated by comparing cell viability after irradiation on nutrient agar and on minimal medium containing either a low or high inorganic salt concentration. An action spectrum for lethality and for cell membrane damage was then determined. From 254 to 310 nm lethality closely corresponded to the absorption spectrum of DNA, and there was no indication of membrane damage. However, above a wavelength of 310 nm, the direct absorption of radiation by DNA could not account for the sensitivity observed. Moreover, at wavelengths longer than 310 nm, cell membrane damage was induced and by an increasing factor up to a peak at 334 nm. At the longer wavelengths of 365 and 405 nm, there was a gradual decrease from the peak of damage to cell membranes induced by 334 nm radiation. These results indicate that cell membrane damage may contribute significantly to near‐UV radiation‐induced cell lethality in wild‐type E. coli K‐12.