A highly sensitive and selective radioimmunoassay for the measurement of neurotensin.

A highly selective and sensitive radioimmunoassay (RIA) for the detection of endogenous neurotensin (NT) has been developed. We have raised a C-terminally-directed antibody (CAb) that specifically binds 'biologically active' NT (NT and NT(8-13)) and that does not significantly cross-react with inactive NT metabolites or other bioactive peptides in the CNS. By reducing the volume of the assay to a low volume-RIA (30 microl), such that in vivo measurements can be made, we have increased the sensitivity (<0.3 fmol per tube), with inter- and intra-assay variations of 11.2 and 5.8%, respectively. Comparisons with similar methods of detecting NT have demonstrated that this RIA has a higher sensitivity than previously used RIA's and ELISA's. The data presented suggests that this sensitive RIA is a reliable method ideal for the detection of small quantities of biologically active NT.

Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes.

Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.

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

PURPOSE: 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.

Developmental changes in neurotensin and its metabolites in the neonatal rat.

Neurotensin-like immunoreactivity (NT-LI) was measured in the di-, tel- and mesencephalon of rats from embryonic day 15 (E15) through birth ( approximately E22) until postnatal day 5 (P5) using radioimmunoassay (RIA) and an N-terminal directed polyclonal antibody. NT-LI and NT metabolite-like immunoreactivities (NT 1-8, NT 1-10, NT 1-11 and NT 1-12-LI) were also similarly determined using high performance liquid chromatography (HPLC) coupled with RIA. NT-LI was low at E15 but increased to peak levels at around E20 or birth in the di- and telencephalon, after which the levels declined. Similar, but lower, changes were observed with NT 1-10-LI but not other metabolites while much lower NT-LI and metabolites were observed in the mesencephalon where no transitory changes occurred. The changes in neonatal rat brain NT and metabolites are discussed with respect to the possible neonatal trophic roles of these peptides.

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.

Binding and biological activity of C-terminally modified melanocortin peptides: a comparison between their actions at rodent MC1 and MC3 receptors.

Five subtypes of melanocortin receptors have to date been identified, but to date little is known about the different structural requirements for binding and biological activity at these receptors. In this study, the role of C-terminal melanocortin peptide residues in imparting selectivity for the receptor subtypes was examined. C-terminally modified analogues of alpha-MSH and gamma-MSH were synthesized and their interaction with MC1 and MC3 melanocortin receptors was investigated. This study provides further evidence for an important role of proline 12 (numbering with respect to alpha-MSH) for binding and activity at the MC1 receptor. Although the influence of C-terminal amino acids on binding and activity at MC3-R was less marked, some of them were nevertheless observed to be beneficial for the interaction with this receptor subtype.

Melanocortin probes for the melanoma MC1 receptor: synthesis, receptor binding and biological activity.

The fate of alpha-melanocyte-stimulating hormone (alpha-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as iodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4,Dphe7]alpha-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.

Receptor binding affinities and biological activities of linear and cyclic melanocortins in B16 murine melanoma cells expressing the native MC1 receptor.

Cyclic alpha-melanocyte-stimulating hormone (alpha-MSH) analogues produced by disulphide bridging (e.g. [Cys4,Cys10] alpha-MSH) are known to be almost equipotent to the native hormone in amphibian skin bioassays and as a consequence have been proposed as a paradigm for the active conformation of native MSH at the pigment cell MC1 receptor. However this proposal has been somewhat speculative as there is no published data comparing biological activity of cyclic MSH analogues with data on receptor binding. This study addresses this problem by comparing tyrosinase stimulatory activity with their receptor binding affinity in B16 murine melanoma cells expressing the native MC1 melanocortin receptor. Cyclic [Cys4,Cys10] alpha-MSH showed almost the same affinity for the MC1 receptor as alpha-MSH, but the linear analogue [Cys4,Cys10] alpha-MSH bound less strongly. Both had biological activities similar to that of the natural ligand. Introduction of D-Phe into the ring in position 7 increased both affinity and activity of the cyclic compound. The study suggests that the intrinsic efficacy of cyclic [Cys4,Cys10] alpha-MSH analogues is similar to native alpha-MSH. Our studies support the proposal that the cyclic structure serves as a good model for the active conformation of linear alpha-MSH.

Molecular modelling of beta turns in a cyclic melanotropin.

Alpha-Melanocyte stimulating hormone (alpha-MSH) is a tridecapeptide which interacts with a family of G protein-coupled receptors, the melanocortin receptors, to cause its biological effects. We have modelled the low energy conformations of the alpha-MSH derivatives as part of a project to probe the receptor binding conformation of melanocortins, and also to design ligands for targeting cytotoxic drugs to MC1 receptors expressed by melanoma cells. Here we report a molecular dynamics study of beta turns in a cyclic lactam analogue [Nle4, Asp5, D-Phe7, Lys10]alpha-MSH. The data show that it is possible for a beta turn to exist in the ring portion of this molecule which contains the melanocortin conserved sequence - His-Phe-Arg-Trp-, even though the lowest energy conformers lack a beta turn.

Identification of a melanocortin receptor expressed by murine brain microvascular endothelial cells in culture.

Microvascular endothelial cells were isolated from the brains of C57 mice and cultured in selective growth media. The isolation and culture techniques employed in this study minimised the contamination by nonendothelial cells such as astrocytes, pericytes, and smooth muscle cells. Microvascular endothelial cells examined using phase contrast light microscopy grew as small colonies of spindle-shaped cells which merged together to form typical contact-inhibited monolayers. The endothelial origin of these cells was determined using several established characterisation techniques. Preliminary receptor binding studies at 4 degrees using [125I-Tyr2, Nle4, D-Phe7]alpha-melanocyte-stimulating hormone ([125I-Tyr2, Nle4, D-Phe7]alpha-MSH) suggested the possibility that melanocortin receptors were present on the surface of brain microvascular endothelial cells. Subsequent binding isotherms confirmed that a small population of high-affinity melanocortin receptors was expressed. The existence of a specific binding site for alpha-MSH was confirmed by photoaffinity labeling with the 4-(1-azi-2,2,2,-trifluoroethyl)benzoic acid (ATB) derivative, [125I-Tyr2, Nle4, D-Phe7, (ATB)-Lys11] alpha-MSH. SDS-PAGE analysis identified the presence of a specific band with a molecular mass of approximately 45 kDa, which was consistent with previous data on melanoma melanocortin receptors, and represented a ligand-receptor complex. This study suggests that a receptor for alpha-MSH is expressed on the extracellular surface of murine brain microvascular endothelial cells; however, the physiological role of this receptor is as yet unknown.

Evidence of photoenzymatic repair due to the phrA gene in a phrB mutant of Escherichia coli K-12.

The role of the phrA gene in the genetic control of photoreactivation in Escherichia coli has been a matter of some controversy. It has been proposed that the gene has no significant physiological role in photoreactivation. However, we have previously sequenced a restriction fragment thought to contain the phrA gene and shown it to contain a putative gene. When this gene, termed the putative phrA gene, was transformed into a phrAphrB mutant, a photoreactivable response above that of the phrAphrB mutant was observed. It has been suggested that the photorepair seen in phrB mutants is due to Type III photoreactivation, which is independent of temperature and fluence rate effects. Here we have shown that the photorecovery associated with the phrA gene is dependent on both temperature and fluence rate. This suggests that the photorecovery is not due to Type III photoreactivation but to an enzymatic reaction caused by an unknown photoactive protein, the phrA gene product, which acts on lesions other than pyrimidine dimers, possibly pyrimidine (6-4) pyrimidone photoproducts. We therefore propose that the phrA gene be reaccepted and its role in photoreactivation in Escherichia coli acknowledged.

The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues.

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-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 alpha-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.

Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine.

The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-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 alpha-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 Met4 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 Phe7, Arg8, and Trp9 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.

Influence of alpha-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.

Photoreactivation in a phrB mutant of Escherichia coli K-12: evidence for the role of a second protein in photorepair.

In Escherichia coli, the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr. This gene was earlier described as phrB, another locus phrA having been proposed in association with PR. The relevance of the putative phrA gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosophila melanogaster, which specifically repairs pyrimidine (6-4) pyrimidone photoproducts ([6-4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6-4) photoproduct, has cast new light on the prospective role of phrA in PR. We have determined the nucleotide sequence of the putative phrA gene, which suggests it codes for a protein of 38 kDa. When the putative phrA gene was cloned into an expression vector and transformed into a phrA phrB mutant of E. coli, a level of photorepair was observed, which could correspond to repair of (6-4) photoproducts.

Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads.

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

Photoreactivation in phr mutants of Escherichia coli K-12: cloning from the gal-att lambda interval increases the photoreactivable response in phrA strains.

In this report we have cloned restriction fragments from the gal-att lambda region obtained from a purified preparation of lambda dgal transducing 'phage DNA, and demonstrate the appearance of a photoreactivable response in a photoreactivation-deficient phrA phrB strain. We also show that when this plasmid is transduced into a delta phrA strain there is an increase in the photoreactivable response after a single high intensity light flash and after continuous illumination. These data have been discussed in relation to the hypothesis of the presence of multiple photolyase molecules in Escherichia coli.

Effect of growth temperature on lipid composition and ultraviolet sensitivity of human cells.

Human skin fibroblasts were incubated at either 25 or 37 degrees C before UV irradiation. Cells incubated at 25 degrees C were more resistant to near UV radiation than cells grown at 37 degrees C, but cells grown at the lower temperature were more sensitive to 254 nm radiation. Fatty acid analysis of membranes of cells showed that cells incubated at the lower temperature contained significantly higher amounts of linoleic acid (18:2) and linolenic acid (18:3) than cells incubated at 37 degrees C. To determine if this difference in fatty acid content of the membranes was responsible for the UV survival characteristics of cells incubated at different temperatures, cells were enriched with either linoleate or linolenate during a 37 degrees C incubation period. Gas chromatography revealed that cells incorporated the supplied fatty acid. Fatty acid enriched cells were then irradiated with near UV, and survival characteristics were compared to those obtained with cells grown at the lower incubation temperature. The results suggest that the different proportion of fatty acid content of the cells is not the cause of different UV sensitivities of cells grown at 25 degrees C compared to cells grown at 37 degrees C.