A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal growth factor receptor in A431 cells

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense mechanism affecting receptor tyrosine kinase activity.

ROS as signalling molecules in T cells - Evidence for abnormal redox signalling in the autoimmune disease, rheumatoid arthritis

Reactive oxygen species are recognised as important signalling molecules within cells of the immune system. This is, at least in part, due to the reversible activation of kinases, phosphatases and transcription factors by modification of critical thiol residues. However, in the chronic inflammatory disease rheumatoid arthritis, cells of the immune system are exposed to increased levels of oxidative stress and the T cell becomes refractory to growth and death stimuli. This contributes to the perpetuation of the immune response. As many of the effective therapies used in the treatment of rheumatoid arthritis modulate intracellular redox state, this raises the question of whether increased oxidative stress is causative of T-cell hyporesponsiveness. To address this hypothesis, this review considers the putative sources of ROS involved in normal intracellular signalling in T cells and the evidence in support of abnormal ROS fluxes contributing to T-cell hyporesponsiveness. © W. S. Maney & Son Ltd.

The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Pro-inflammatory cytokines IL6, TNF-α, IL1β, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis

Objective: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). Patients and methods: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-α (TNF-α), interleukin 1-β (IL1β), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Results: Serum levels of IL6, IL1β and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-α and LBP were not elevated. Conclusion: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome. © 2007 The British Infection Society.

Propionibacterium acnes

Propionibacterium acnes, a common skin organism, is most notably recognized for its role in acne vulgaris. It also causes postoperative and device-related infections and has been associated with a number of other conditions such as sarcoidosis and synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO), although its precise role as a causative agent remains to be determined. Propionibacterium acnes produces a number of virulence factors and is well known for its inflammatory and immunomodulatory properties. Recent publication of the P. acnes genome should provide further insights into the pathogenic capabilities of the organism and potentially lead to the development of new therapies. © 2006 The Society for Applied Microbiology.

Evaluation of PCR in the molecular diagnosis of endocarditis

Objective. Infective endocarditis (IE) is diagnosed by the Duke criteria, which can be inconclusive particularly when blood cultures are negative. This study investigated the application of polymerase chain reaction (PCR) to identify bacterial DNA in excised valvular tissue, and its role in establishing the diagnosis of IE. Methods. Ninety-eight patients undergoing valve replacement surgery were studied. Twenty-eight patients were confirmed as definite for endocarditis by the Duke criteria; nine were considered as possible and 61 had no known or previous microbial infection of the endocardium. A broad-range PCR technique was used to amplify prokaryotic 16S rRNA genes present within homogenised heart valve tissue. Subsequent DNA sequencing of the PCR amplicon allowed identification of the infecting microorganism. Results. PCR results demonstrated the presence of bacterial DNA in the heart valves obtained from 14 out of 20 (70%) definite IE patients with positive blood cultures preoperatively. The causative microorganism for one patient with definite culture negative endocarditis was identified by PCR. Two out of nine (22%) of the valves from possible endocarditis patients also had bacterial DNA present converting them into the definite criteria whereas in the valves of seven out of nine (78%) of these patients no bacterial DNA was detected. Conclusion. The application of PCR to the explanted valves in patients with possible or confirmed diagnosis can augment the Duke criteria thereby improving post-surgical antimicrobial therapeutic options. © 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Comparative performance of analytical techniques for polyhexamethylene biguanide (PHMB) in ophthalmic solutions

Purpose: The use of PHMB as a disinfectant in contact lens multipurpose solutions has been at the centre of much debate in recent times, particularly in relation to the issue of solution induced corneal staining. Clinical studies have been carried out which suggest different effects with individual contact lens materials used in combination with specific PHMB containing care regimes. There does not appear to be, however, a reliable analytical technique that would detect and quantify with any degree of accuracy the specific levels of PHMB that are taken up and released from individual solutions by the various contact lens materials. Methods: PHMB is a mixture of positively charged polymer units of varying molecular weight that has maximum absorbance wavelength of 236 nm. On the basis of these properties a range of assays including capillary electrophoresis, HPLC, a nickelnioxime colorimetric technique, mass spectrophotometry, UV spectroscopy and ion chromatography were assessed paying particular attention to each of their constraints and detection levels. Particular interest was focused on the relative advantage of contactless conductivity compared to UV and mass spectrometry detection in capillary electrophoresis (CE). This study provides an overview of the comparative performance of these techniques. Results: The UV absorbance of PHMB solutions, ranging from 0.0625 to 50 ppm was measured at 236 nm. Within this range the calibration curve appears to be linear however, absorption values below 1 ppm (0.0001%) were extremely difficult to reproduce. The concentration of PHMB in solutions is in the range of 0.0002–0.00005% and our investigations suggest that levels of PHMB below 0.0001% (levels encountered in uptake and release studies) can not be accurately estimated, in particular when analysing complex lens care solutions which can contain competitively absorbing, and thus interfering, species in the solution. The use of separative methodologies, such as CE using UV detection alone is similarly limited. Alternative techniques including contactless conductivity detection offer greater discrimination in complex solutions together with the opportunity for dual channel detection. Preliminary results achieved by TraceDec1 contactless conductivity detection, (Gain 150%, Offset 150) in conjunction with the Agilent capillary electrophoresis system using a bare fused silica capillary (extended light path, 50 mid, total length 64.5 cm, effective length 56 cm) and a cationic buffer at pH 3.2, exhibit great potential with reproducible PHMB split peaks. Conclusions: PHMB-based solutions are commonly associated with the potential to invoke corneal staining in combination with certain contact lens materials. However this terminology ‘PHMBbased solution’ is used primarily because PHMB itself has yet to be adequately implicated as the causative agent of the staining and compromised corneal cell integrity. The lack of well characterised adequately sensitive assays, coupled with the range of additional components that characterise individual care solutions pose a major barrier to the investigation of PHMB interactions in the lenswearing eye.

Developments in assessment and teaching methodologies in modern languages in England and France in response to educational reform (1975-1985)

This comparative study considers the main causative factors for change in recent years in the teaching of modern languages in England and France and seeks to contribute, in a general sense, to the understanding of change in comparable institutions. In England by 1975 the teaching of modern languages in the comprehensive schools was seen to be inappropriate to the needs of children of the whole ability-range. A combination of the external factor of the Council of Europe initiative in devising a needs-based learning approach for adult learners, and the internal factor of teacher-based initiatives in developing a graded-objectives learning approach for the less-able, has reversed this situation to some extent. The study examines and evaluates this reversal, and, in addition, assesses teachers' attitudes towards, and understanding of, the changes involved. In France the imposition of `la reforme Haby' in 1977 and the creation of `le college unique' were the main external factors for change. The subsequent failure of the reform and the socialist government's support of decentralisation policies returning the initiative for renewal to schools are examined and evaluated, as are the internal factors for changes in language-teaching - `groupes de niveau' and the creation of `equipes pedagogiques'. In both countries changes in the function of examinations at 15/16 plus are examined. The final chapter compared the changes in both education systems.

The interaction between master production scheduling and detailed capacity requirements planning : closing the loop in manufacturing resource planning (MRPII) systems

The widespread implementation of Manufacturing Resource Planning (MRPII) systems in this country and abroad and the reported dissatisfaction with their use formed the initial basis of this piece of research which concentrates on the fundamental theory and design of the Closed Loop MRPII system itself. The dissertation concentrates on two key aspects namely; how Master Production Scheduling is carried out in differing business environments and how well the `closing of the loop' operates by checking the capcity requirements of the different levels of plans within an organisation. The main hypothesis which is tested is that in U.K. manufacturing industry, resource checks are either not being carried out satisfactorily or they are not being fed back to the appropriate plan in a timely fashion. The research methodology employed involved initial detailed investigations into Master Scheduling and capacity planning in eight diverse manufacturing companies. This was followed by a nationwide survey of users in 349 companies, a survey of all the major suppliers of Production Management software in the U.K. and an analysis of the facilities offered by current software packages. The main conclusion which is drawn is that the hypothesis is proved in the majority of companies in that only just over 50% of companies are attempting Resource and Capacity Planning and only 20% are successfully feeding back CRP information to `close the loop'. Various causative factors are put forward and remedies are suggested.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

A study of the calcium-induced morphological transistions of human erythrocytes

An examination was made of the morphological transitions induced in human erythrocytes by the elevation of cytosolic calcium, and of the biochemical mechanisms responsible. The loss of the discocyte morphology and the sequential progression of cells through the echinocyte stages 1, 2, 3 and sphereo-echinocyte was found to occur in both a calcium concentration- and a time-dependent manner. SDS-PAGE analysis of cytoskeletal proteins prepared from intact cells loaded with 150uM or 1mM calcium revealed the partial proteolytic loss of proteins 2.1, 2.2 and 4.1. The rate of proteolysis was not paralleled by that of echinocytosis, making a causative relationship unlikely. Cytoskeletal integrity did appear to influence shape reversal from the echinocyte to the discocyte morphology after removal of the calcium and ionophore A23187. The loss of 80% protein 4.1, 40% 2.1 and 30% 2.2 was associated with, although not necessarily the sole cause, of irreversible sphereo-echinocytosis. Pre-treatment of cells with wheat germ agglutinin preserved the discocyte morphology despite continued cytoskeletal proteolysis during calcium-loading. All observations were made on cells incubated either in the presence or absence of glycolytic substrates, effectively altering cell metabolic status. This influenced the rate of progression of cells through the echinocyte stages, the rate of proteolysis of cytoskeletal proteins, and the extent and kinetics of shape reversal from cells transformed to the sphereo-echinocyte morphology. The stage 1 to discocyte transition was the rate limiting step of this shape recovery. In contrast the rate of loss of the discocyte morphology was independent of cell metabolic status during exposure to calcium, as was the extent of restoration of the discocyte morphology from cells transformed to stage 1 echinocytes. An hypothesis is presented that echinocytosis is a discontinuous process with discrete steps initiated by different biochemical mechanisms varying in their dependence on metabolic energy.

Ophthalmic factors in dyslexia

Although the role of ophthalmic factors in dyslexia remains the subject of controversy, recent research has indicated that the correlates of dyslexia may include binocular dysfunction, unstable motor ocular dominance, a deficit of the transient visual subsystem, and an anomaly that can be treated with tinted lenses. These features, typically, have been studied in isolation and their inter-relationship has received little attention. The aim of the present research was to investigate ophthalmic factors in dyslexia, with a particular emphasis on the interaction between optometric variables. Further aims were to establish the most appropriate investigative techniques for optometric practice and to explore the relationship between optometric and psychometric variables. A pilot study was used to refine the experimental design for a subsequent detailed study of 39 children with a specific reading disability and 43 good readers, who were selected from 240 children. The groups were matched for age, sex, and performance IQ. The following factors emerged as correlates of dyslexia: slight impaired visual acuity; reduced vergence amplitudes; increased vergence instability; decreased accommodative amplitude; poor peformance at tests that were designed to assess the function of the transient visual system; and slightly slower performance at a non-verbal simulated reading visual search task. The `transient system deficit', as measured by reduced flicker sensitivity, was significantly associated with decreased accommodative and vergence amplitudes. This links the motor and sensory visual correlates of dyslexia. Although the binocular dysfunction was correlated with increased symptoms, the difference in the groups' simulated reading visual search task performance was largely attributable to psychometric variables. The results suggest tht optometric problems may be a contributory factor in dyslexia, but are unlikely to play a key causative role. Several optometric variables were confounded by psychometric parameters, and this interaction should be a priority for future investigation.

Cancer cachexia

Causative factors: Nutritional supplementation or pharmacological manipulation of appetite are unable to control the muscle atrophy seen in cancer cachexia. This suggests that tumour and/or host factors might be responsible for the depression in protein synthesis and the increase in protein degradation. An increased expression of the ubiquitin-proteasome proteolytic pathway is responsible for the increased degradation of myofibrillar proteins in skeletal muscle, and this may be due to tumour factors, such as proteolysis-inducing factor (PIF), or host factors such as tumour necrosis factor-α (TNF-α). In humans loss of adipose tissue is due to an increase in lipolysis rather than a decrease in synthesis, and this may be due to tumour factors such as lipid-mobilising factor (LMF) or TNF-α, both of which can increase cyclic AMP in adipocytes, leading to activation of hormone-sensitive lipase (HSL). Levels of mRNA for HSL are elevated twofold in adipose tissue of cancer patients, while there are no changes in lipoprotein lipase (LPL), involved in extraction of fatty acids from plasma lipoproteins for storage. Treatment for cachexia: This has concentrated on increasing food intake, although that alone is unable to reverse the metabolic changes. Agents interfering with TNF-α have not been very successful to date, although more research is required in that area. The only agent tested clinically that is able to interfere with the action of PIF is eicosapentaenoic acid (EPA). EPA attenuates protein degradation in skeletal muscle by preventing the increased expression of the ubiquitin-proteasome pathway, but has no effect on protein synthesis. When used alone EPA prevents further wasting in cachectic patients, and, when it is combined with an energy- and protein-dense nutritional supplement, weight gain is seen, which is totally lean body mass. These results suggest that mechanistic studies into the causes of cancer cachexia will allow appropriate therapeutic intervention.

VaxiJen:a server for prediction of protective antigens, tumour antigens and subunit vaccines

Background - Vaccine development in the post-genomic era often begins with the in silico screening of genome information, with the most probable protective antigens being predicted rather than requiring causative microorganisms to be grown. Despite the obvious advantages of this approach – such as speed and cost efficiency – its success remains dependent on the accuracy of antigen prediction. Most approaches use sequence alignment to identify antigens. This is problematic for several reasons. Some proteins lack obvious sequence similarity, although they may share similar structures and biological properties. The antigenicity of a sequence may be encoded in a subtle and recondite manner not amendable to direct identification by sequence alignment. The discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. To overcome the limitations of alignment-dependent methods, we propose a new alignment-free approach for antigen prediction, which is based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties. Results - Bacterial, viral and tumour protein datasets were used to derive models for prediction of whole protein antigenicity. Every set consisted of 100 known antigens and 100 non-antigens. The derived models were tested by internal leave-one-out cross-validation and external validation using test sets. An additional five training sets for each class of antigens were used to test the stability of the discrimination between antigens and non-antigens. The models performed well in both validations showing prediction accuracy of 70% to 89%. The models were implemented in a server, which we call VaxiJen. Conclusion - VaxiJen is the first server for alignment-independent prediction of protective antigens. It was developed to allow antigen classification solely based on the physicochemical properties of proteins without recourse to sequence alignment. The server can be used on its own or in combination with alignment-based prediction methods.

Resveratrol inhibits the release of soluble fms-like tyrosine kinase (sFlt-1) from human placenta

Objective - Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release. Study Design - Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting. Results - Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia. Conclusion - In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.