ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival.

The ATP-gated receptor P2X7 is expressed in multiple malignant tumours including neuroblastoma, melanoma, prostate, lung and breast. P2X7 has a significant role in mediating diverse cell responses, which upon dysregulation are associated with tumour initiation and development. The rapid, ATP-mediated activation of P2X7 induces a fast-inward cation current in cells. However, prolonged ATP-mediated activation of P2X7 leads to formation of a pore that increases membrane permeability and eventually causes cell death. This presents a potential paradox, as the tumour microenvironment contains extracellular ATP at levels sufficient to activate the P2X7 pore and trigger cell death. However, P2X7 expression is associated with enhanced cancer cell survival, proliferation and metastatic potential. At least one distinct conformational form of P2X7, termed non-pore functional P2X7 (nfP2X7), has been described, which is not able to form a functional pore. We demonstrate for the first time in this study that exposure to a high ATP concentration, equivalent to those measured in the tumour microenvironment, drives nfP2X7 expression and also that nfP2X7 is essential for tumour cell survival. We show that monoclonal antibodies raised against a P2X7 amino acid sequence (200-216), whose conformation is distinct from that of wild-type (WT) P2X7, bind specifically to nfP2X7 expressed on the surface of tumour cells. We also show that nfP2X7 is broadly expressed in patient-derived tumour sections from a wide range of cancers. Therefore, antibodies raised against E200 provide tools that can differentiate between forms of the P2X7 receptor that have a key role in cancer.

A phase I clinical trial demonstrates that nfP2X7 -targeted antibodies provide a novel, safe and tolerable topical therapy for basal cell carcinoma.

BACKGROUND: Expression of P2X7 , an ATP-gated calcium channel, increases cancer cell proliferation and invasiveness. A variant of P2X7 (termed nfP2X7 ), in which a normally hidden epitope (E200) is exposed for antibody binding, is observed in a variety of different cancers. OBJECTIVES: To investigate the safety, tolerability and pharmacokinetics and assess indicative efficacy of a novel antibody ointment as a therapeutic for basal cell carcinoma (BCC). METHODS: An open-label, phase I clinical trial was undertaken at three dermatology clinics to evaluate the safety and tolerability of topical administration of an ointment containing 10% sheep polyclonal anti-nfP2X7 antibodies (BIL010t) to primary BCC lesions twice daily for 28 days. Twenty-one patients with primary BCC lesions at least 0·5 cm2 in area and less than 2·0 cm in diameter were enrolled. The primary end points were safety, tolerability and pharmacokinetics. Change in lesion size after treatment was determined and histology was performed on pretreatment and end-of-treatment (EOT) biopsies. RESULTS: Compliance was very high, with treatment being well tolerated. The most common adverse events were treatment site erythema, pruritus, dryness and pain. There was no evidence of systemic penetration of the sheep antibody. Lesions were measured prior to and after 28 days of treatment, with 65% of patients showing a reduction in lesion area, 20% showing no change and 15% showing an increase. Histopathology of post-treatment excision of lesion sites showed eight patients with stable disease, nine with partial response and three with complete response. CONCLUSIONS: Antibodies against nfP2X7 (BIL010t) provide a novel, safe and well-tolerated treatment for BCC.

Molecular structures and interactions of repetitive peptides based on wheat glutenin subunits depend on chain length.

Synthetic and recombinant peptide models of the central repetitive domain of the high molecular weight subunits of wheat glutenin with different numbers of the consensus repeat motifs PGQGQQ + GYYPTSLQQ (21, 45, 110, and 203 residues long) and a recombinant 58,000-Da relative molecular mass (M(r) 58,000) repetitive peptide from a single subunit (1Dx5) are studied using Fourier transform IR spectroscopy. The spectra of the dry peptides are very similar; at low water contents (<76% relative humidity) there is an increase in beta-sheet structures in all peptides. However, on further hydration the content of beta sheets decrease and more beta turns are observed. The changes during the second step of hydration are very marked in the 21 and 45 residue peptides, but they are less apparent in the longer perfect repeat peptides. In the 110 and 203 residue peptides hydration results in increased contents of intermolecular beta-sheets and less beta-turn formation. In contrast, the beta-turn content of the M(r) 58,000 peptide increases during the second hydration step. The decreased extent of structure changes with increasing chain length indicates that cumulative intermolecular interactions, in particular hydrogen bonds, are an important factor in determining the structures in the solid state. The regularity of the perfect repeat sequences in the 21, 45, 110, and 203 residue peptides may favor the formation of larger stretches of intermolecular beta sheets. In contrast, the M(r) 58,000 peptide contains imperfect repeats (in common with native glutenin subunits), which may limit its ability to form intermolecular beta sheets.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Interaction between protein allergens and model gastric emulsions.

The observed resistance to pepsinolysis of known food allergens has been suggested as a predictor of their allergenic risk. Consequently, resistance to pepsinolysis has become incorporated into decision tree assessment for potential allergenic risk posed by novel foods. However, existing methods take little account of the interaction between food structure and physiological conditions existing during digestion in vivo. Here we show that a range of protein allergens can adsorb to model stomach emulsions, providing a further means of resisting digestion. We also show that raising the pH and the addition of bile salts to a model stomach emulsion, thereby mimicking the duodenal environment, has the effect of desorbing the adsorbed protein.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.

A high resolution (1)H magic angle spinning NMR study of a high-M(r) subunit of wheat glutenin.

This work describes the application of (1)H magic angle spinning (MAS) nmr to the study of hydrated 1Dx5 wheat high-M(r) subunit. 1Dx5 is a water-insoluble 88 kDa protein, associated with good baking performance, and whose structure in the solid and low-hydration states is not known. High-resolution MAS (HR-MAS) results in a threefold resolution improvement of the (1)H spectra of the hydrated wheat protein, compared to standard MAS. The spectral resolution achieved enables, for the first time, two-dimensional nmr methods to be employed for the study of hydrated 1Dx5 and the assignment of the spectrum to be carried out on the basis of total correlated spectroscopy and (13)C/(1)H correlation experiments. Considerable shifts are observed for some resonances, relative to the chemical shifts of amino acids in solution, indicating that specific interactions occur in the hydrated protein network. Two main environments are identified for glutamine residues, Q(1) and Q(2), and these were characterized in terms of possible conformation and relative dynamics, with the basis of comparison between the single 90 degrees spectrum and the Carr-Purcel-Heiboom-Gill (CPMG) spectrum. The Q(1) residues are proposed to be situated in protein segments that adopt the beta-sheet conformation and that remain relatively hindered, possibly by hydrogen bonds involving the glutamine amide groups. On the other hand, Q(2) residues are proposed to be situated in a more mobile environment, adopting a looser conformation, possibly a beta-turn conformation. Based on the proximity of the Q(2) residues with glycine residues, as viewed by the nuclear Overhauser effect spectroscopy experiment, it is proposed that the protein segments that form the more mobile (or loop) sections of the network are rich in both glutamine and glycine residues.

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein.

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.

Extraction, separation, and purification of wheat gluten proteins and related proteins of barley, rye, and oats.

The wheat proteins, which are active in celiac disease and other glutenrelated conditions, are defined as prolamins, in that they are soluble as individual subunits in alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60-70% (v/v) aqueous ethanol. However, in wheat grain and flour, about half of these subunits are present in polymers that are not soluble in alcohol-water mixtures unless disulfide bonds between the component subunits are reduced using an agent such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). These alcohol-insoluble polymers are traditionally called glutenins and the related alcohol-soluble monomers are called gliadins; the two groups of proteins together form the major part of the gluten fraction. Gluten can be readily prepared from wheat by washing dough to remove the bulk of the starch, cell-wall material, and soluble components. It is a cohesive viscoelastic mass that contains, in addition to the gluten proteins, small amounts of other proteins, residual starch (about 25% dry wt), and lipid.

Correlation of response to treatment and HIV genotypic changes during phase III trials with saquinavir and reverse transcriptase inhibitor combination therapy.

OBJECTIVES: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment. DESIGN: Virologic substudies of Phase III clinical trials NV14256 and SV14604. METHODS: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates. RESULTS: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV. CONCLUSIONS: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.

Therapeutic efficacy and bone mineral density response during and following a three-month re-treatment of endometriosis with nafarelin (Synarel).

OBJECTIVE: Our goal was to determine the effects of a repeated course of the gonadotropin-releasing hormone agonist nafarelin on symptoms and signs of endometriosis and lumbar and distal radius bone mineral density. STUDY DESIGN: Forty-five women previously treated for 6 months with nafarelin, who had recurrent symptoms and signs of endometriosis, received 400 mcg/day of nafarelin intranasally for 3 months. Efficacy was evaluated by changes in severity of symptoms and signs. Lumbar bone mineral density was measured by dual-energy x-ray absorptiometry and distal radius bone mineral density by single-photon absorptiometry. Bone mineral density was also measured in 10 control volunteers. RESULTS: Repeated 3-month treatment significantly alleviated recurrent symptoms and signs of endometriosis. Lumbar bone mineral density decreased significantly by a mean of 2% at the end of treatment; this loss was restored within 3 to 6 months after treatment completion. No bone mineral density decline occurred in the radius. Bone mineral density changes in the control group were statistically insignificant. CONCLUSIONS: A repeated 3-month course of nafarelin treatment significantly relieved recurrent endometriotic symptoms and signs without sustained loss of bone mineral density.

SO42- Deprivation Has an Early Effect on the Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Photosynthesis in Young Leaves of Wheat.

Wheat (Triticum aestivum cv Chinese Spring) supplied with 0.45 mM SO42- for 14 d with relative growth rates (RGR) of 0.22 to 0.24 d-1 was deprived of S for 7 to 8 d. There was no significant effect on RGR or leaf development (leaf 2 length was constant; leaf 3 expanded for 2-4 d; leaf 4 emerged and elongated throughout the experiment) during the S deprivation. In controls the net assimilation rate (A) closely reflected leaf ontogeny. S deprivation affected A in all leaves, particularly leaf 4, in which A remained at 8 to 10 [mu]mol CO2 m-2 s-1, whereas in controls A rose steadily to >20 [mu]mol CO2 m-2 s-1. In leaf 2, with a fully assembled photosynthetic system, A decreased in S-deprived plants relative to controls only at the end of the experiment. Effects on A were not due to altered stomatal conductance or leaf internal [CO2] ([C]i); decreases in the initial slope of A/[C]i curves indicated an effect of S deprivation on the carboxylase efficiency. Measurement of Rubisco activity and large subunit protein abundance paralleled effects on A and A/[C]i in S-deprived leaves. Negative effects on photosynthesis in S-deprived plants are discussed in relation to mobilization of S reserves, including Rubisco, emphasizing the need for continuous S supply during vegetative growth.

Expression of genes involved in phycocyanin biosynthesis following recovery of Synechococcus PCC 6301 from nitrogen starvation, and the effect of gabaculine on cpcBa transcript levels.

The effect of the tetrapyrrole biosynthesis inhibitor gabaculine on the expression of specific genes involved in phycocyanin biosynthesis was investigated in cultures of Synechococcus PCC6301 in nitrogen chlorosis, and during recovery to nitrogen sufficiency. Patterns of transcription of the cpcBA (phycocyanin subunits), hemL (glutamate semialdehyde aminotransferase) and hemB (aminolaevulinate dehydratase) genes were visualised by Northern blotting and gene product formation for cpcBA, hemL and the gene for glu tRNA synthetase were monitored by Western blotting. Inhibition of phycobilin biosynthesis by gabaculine greatly decreased production of phycocyanin protein and of cpcBA transcript, indicating a tight coordination of apoprotein biosynthesis with chromophore supply at the level of transcription. Different patterns of response were observed with the other genes at the level of transcript formation or gene product synthesis.

Q-switched neodymium-YAG laser iridotomy. A field trial with a portable laser system.

The efficacy of a small, portable, battery-operated, Q-switched neodymium-YAG laser with a slit-lamp delivery system was evaluated in a short-term pilot study. Iridotomies were created in 44 Eskimo eyes (23 patients) with occludable angles in Alaska's Kotzebue region. The laser was transported as regular baggage, was used in three villages (utilizing available facilities), and was operational within five minutes. Patent iridotomies were achieved in all eyes and with one pulse in 18 eyes (44%). Complications included transient bleeding from the iridotomy site in 23 eyes (52%), focal corneal opacities in 11 eyes (25%), and a transient immediate postoperative intraocular pressure elevation in nine eyes (20%). This appears to be the first portable laser system that can be used in frontier areas and underdeveloped nations to prophylactically treat pupillary-block glaucoma.