Proteomic identification of secreted proteins as surrogate markers for signal transduction inhibitor activity.

Epidermal growth factor receptor is a potential target for cancer treatment and new small-molecule tyrosine kinase inhibitor drugs have been designed to inhibit its activity. In this work we identify potential surrogate markers of drug activity using a proteomic analysis. Two-dimensional electrophoresis was optimised to compare expression patterns of proteins secreted from the cancer cell lines A431 and A549 treated with Gefitinib (Iressa) vs untreated or vehicle-only-treated samples. Upregulated or downregulated proteins were detected using Phoretix 2D image analysis software. Several proteins were then identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. In one case, upregulation of Protein Disulphide Isomerase in response to Gefitinib was confirmed by Western blot analysis, and the response was shown to be concentration dependent. The identification of surrogate markers may be of use for the evaluation of new drugs, in preclinical models, in clinical trials and in the therapy of individual patients to give optimal biological drug doses.

Identification of surrogate markers for determining drug activity using proteomics.

In a high proportion of human carcinomas overexpression of the EGFR (epidermal growth factor receptor), a receptor tyrosine kinase, represents a potential target for cancer treatment. EGFR is induced to dimerize through ligand binding which activates the tyrosine kinase activity of the receptor. This catalyses the transfer of ATP's gamma-phosphate to hydroxyl groups of tyrosine residues on the receptor, creating binding sites that recruit downstream signalling proteins. New drugs, SMTKIs (small-molecule tyrosine kinase inhibitors), have been designed to inhibit the tyrosine kinase activity of the receptor, producing an anti-tumour effect. The development of surrogate markers to determine the drug activity of these new inhibitors would be of great benefit in drug evaluation and in the subsequent management of patient disease. This review describes current treatments of cancer using tyrosine kinase inhibitors and the use of proteomic analysis to identify possible markers of activity of these new drugs.

99mTc-SnF2 colloid "LLK": particle size, morphology and leucocyte labelling behaviour.

99mTc-SnF2 colloid (Radpharm LLK) leucocyte labelling agent is used in whole blood, exploiting phagocytosis. The objectives of this work were to optimize leucocyte labelling in leucocyte-enriched plasma, and to investigate: (i) the effect of temperature and other factors on labelling efficiency; (ii) the selectivity for different leucocyte types; (iii) the viability of the labelled cells and efflux of the radiolabel; and (iv) the physical characteristics of the colloid. Density gradient centrifugation was used to investigate the labelling efficiency, cell selectivity and efflux, Trypan blue to study the viability, and laser scattering, electron microscopy and membrane filtration to investigate particle size and morphology. Particles appeared as loose, coiled, chain-like aggregates of much smaller particles (<0.05 microm). The aggregate diameter ranged from <0.1 to >5 microm and increased with time. The distribution of radioactivity amongst the particle sizes varied widely. The labelling efficiency in leucocyte-rich plasma was enhanced at 37 degrees C compared to room temperature, and by centrifuging during labelling. The selectivity for different leucocyte types varied markedly between batches and blood samples, in some cases showing preference for mononuclear cells and in others for granulocytes. Viability was excellent and comparable with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO)-labelled cells. A significant fraction of radiolabel, comparable to that observed with 99mTc-HMPAO, was lost from leucocytes during incubation in vitro over 4 h. Thus, 99mTc-SnF2 is a convenient, efficient labelling agent for leucocytes, but shows variable cell selectivity which may be linked to particle size variability, and there is significant efflux of radioactivity from labelled cells.

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific.

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.

Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs.

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.

Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease.

This study describes the formulation, characterisation and preliminary clinical evaluation of mucoadhesive, semi-solid formulations containing hydroxyethylcellulose (HEC, 1-5%, w/w), polyvinylpyrrolidine (PVP, 2 or 3%, w/w), polycarbophil (PC, 1 or 3%, w/w) and tetracycline (5%, w/w, as the hydrochloride). Each formulation was characterised in terms of drug release, hardness, compressibility, adhesiveness (using a texture analyser in texture profile analysis mode), syringeability (using a texture analyser in compression mode) and adhesion to a mucin disc (measured as a detachment force using the texture analyser in tensile mode). The release exponent for the formulations ranged from 0.78+/-0.02 to 1. 27+/-0.07, indicating that drug release was non-diffusion controlled. Increasing the concentrations of each polymeric component significantly increased the time required for 10 and 30% release of the original mass of tetracycline, due to both increased viscosity and, additionally, the unique swelling properties of the formulations. Increasing concentrations of each polymeric component also increased the hardness, compressibility, adhesiveness, syringeability and mucoadhesion of the formulations. The effects on product hardness, compressibility and syringeability may be due to increased product viscosity and, hence, increased resistance to compression. Similarly, the effects of these polymers on adhesiveness/mucoadhesion highlight their mucoadhesive nature and, importantly, the effects of polymer state (particularly PC) on these properties. Thus, in formulations where the neutralisation of PC was maximally suppressed, adhesiveness and mucoadhesion were also maximal. Interestingly, statistical interactions were primarily observed between the effects of HEC and PC on drug release, mechanical and mucoadhesive properties. These were explained by the effects of HEC on the physical state of PC, namely swollen or unswollen. In the preliminary clinical evaluation, a formulation was selected that offered an appropriate balance of the above physical properties and contained 3% HEC, 3% PVP and 1% PC, in addition to tetracycline 5% (as the hydrochloride). The clinical efficacy of this (test) formulation was compared to an identical tetracycline-devoid (control) formulation in nine periodontal pockets (>/=5 mm depth). One week following administration of the test formulation, there was a significant improvement in periodontal health as identified by reduced numbers of sub-gingival microbial pathogens. Therefore, it can be concluded that, when used in combination with mechanical plaque removal, the tetracycline-containing semi-solid systems described in this study would augment such therapy by enhancing the removal of pathogens, thus improving periodontal health.

Direct PCR of Cryptococcus neoformans MATalpha and MATa pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies.

Cryptococcus neoformans MATalpha and MATa pheromones were amplified by direct PCR. Nucleotide sequence analyses revealed unique restriction enzyme sites. Sixty strains were used to devise a restriction fragment length polymorphism typing scheme that yielded three variety-specific patterns. Additionally, pheromone-specific PCR allowed easier identification of diploid C. neoformans strains than flow cytometry.

A preliminary study of patient comfort associated with customised mouthguards.

OBJECTIVE: To compare patient perception of custom made mouthguards of ideal and less than ideal designs in terms of their comfort and "wearability". METHOD: A mouthguard of ideal design (A) and one incorporating common design faults of underextension and unadjusted occlusion (B) were provided for 22 active sportsmen and women. They were not informed of the details of the design or the status of the protector. Half the participants were asked to wear mouthguard A first and the other half wore B first, each worn for one hour on two consecutive nights. Questionnaires were used to evaluate and rate the comfort and wearability of each mouthguard. RESULTS: Eighteen people completed the study. The ideal appliance was rated as significantly more retentive and comfortable overall and specifically was more comfortable to lips, gums, and tongue. It was also recognised as being less bulky, less likely to keep the teeth apart, or to cause pain in the jaw muscles. CONCLUSIONS: Comfort is likely to be increased if mouthguards are extended labially to within 2 mm of the vestibular reflection, adjusted to allow even occlusal contact, rounded at the buccal peripheries, and tapered at the palatal edges.

Analysis of lentiviral genomic variation by denaturing gradient gel electrophoresis.

Retroviruses are known for their genetic variability. In any infection, several genotypes usually exist within the host. We have used denaturing gradient gel electrophoresis to study genetic variation of ovine lentiviruses. Starting with viral DNA from cells infected in vitro, a portion of the envelope gene was amplified by PCR, and the products were analyzed by DGGE. With this technique we have been able to detect sequence variations between and within virus isolates and to show evolution of the predominant viral species upon in vivo passage.

A review of 100 consecutive Richard's total knee replacements.

One hundred consecutive Richard's Maximum Contact (RMC) knee replacements were performed in Belfast between 1978 and 1982. Most of the 100 knees (86 patients) involved had been in severe pain, had marked stiffness or gross knee deformity, or were chairbound because of the knee. They were reviewed between five and eight and a half years (mean five years and eleven months) after operation. Thirteen patients (13 knees) died before review leaving eighty seven knees in 73 patients available for study. Using a modification of the British Orthopaedic Association knee function assessment chart, 26 knees (30%) were graded as excellent, 22 (25%) as good, 19 (22%) as fair and nine (10%) as poor. There were five implant failures, four the result of deep infection, one due to loosening. Six patients were chairbound at review and were also graded as failures. These results support the view that total knee replacement approaches the predictability and success of arthroplasty of the hip.

Substituted benzamides with conformationally restricted side chains. 2. Indolizidine derivatives as central dopamine receptor antagonists.

The substituted benzamides metoclopramide (1) and clebopride (3) are stimulants of gastric motility. They are also central dopamine receptor antagonists with 3 being the more potent. This is presumed to be due to an additional interaction of its N-benzyl group with the receptor. The effect of restricting the conformation of this group by replacing the N-benzylpiperidine side chain of 3 by phenyl-substituted quinolizidines and indolizidines has been investigated. Only the indolizidines had significant activity, the nature of which depended upon the orientation of the phenyl substituent. The 2 alpha-phenyl isomers 5d-h were potent central dopamine D2 receptor antagonists with 5h showing selectivity for the limbic system. The 2 beta-phenyl isomer 5c was a gastric motility stimulant devoid of significant central dopamine receptor antagonist activity. Implications on receptor models are discussed.

Population frequencies of carbonic anhydrase II (CA II), esterase D (EsD), and glyoxalase I (GLO) in the Metropolitan Birmingham, Alabama area.

Both black and white populations from Birmingham, Alabama were analyzed for the frequencies of carbonic anhydrase II (CA II), glyoxalase I (GLO) and esterase D (EsD) isoenzymes. The results compared favorably with published frequencies of these genetic markers in other populations.

Increased gastric cholinergic activity evoked by 5-hydroxy-L-tryptophan in the rat.

In gastrointestinal tissues such as rat stomach, exogenous 5-hydroxytryptamine (5HT) has little or no ability to affect nerve activity. However, endogenous 5HT might act differently, and this was investigated by stimulating 5HT synthesis using 5-hydroxy-L-tryptophan (5HTP). In longitudinal strips of rat forestomach, 5HTP (50 and 500 microM) increased cholinergically mediated contractions evoked by electrical field stimulation, probably by facilitating acetylcholine release; contractions evoked by exogenous acetylcholine were unaffected by 5HTP. The ability of 5HTP to increase electrically evoked contractions was long-lasting, required the presence of pyridoxal (a monoamine decarboxylase cofactor) and was reduced by the decarboxylase inhibitor carbidopa, but not by 6-hydroxydopamine. In the presence of paroxetine and nialamide, the 5HTP-induced increase in cholinergically mediated contractions was short-lasting. In anaesthetised rats 5HTP caused stimulation of gastric motility, which was blocked or reduced by atropine. These findings suggest that in the rat 5HTP stimulates gastric cholinergic activity, by increasing the concentration of 5HT at sites which normally synthesise 5HT.

High tibial osteotomy in degenerate diseases of the knee.

Between 1970 and 1981, 64 patients underwent 77 tibial osteotomies for degenerate diseases of the knee at the Withers Orthopaedic Centre in Belfast. Records on 11 patients (12 knees) were either missing or inadequate, leaving 53 patients who underwent 65 tibial osteotomies for study. There were 23 males and 30 females, ranging in age from 23 to 75 years (mean 59.8 years). The predominant diagnosis was osteoarthrosis and the indication for operation in all cases was pain. With a follow-up of from two to ten years (mean 4.8 years), 39 knees were assessed as good, 15 as fair (improved, but still symptomatic), and 11 as failures. Patients with valgus deformity did worse than those with varus deformity. The importance of adequate pre-operative assessment is stressed, the operation itself is outlined, and the end result is seen to correlate closely with the degree of correction obtained.

[Vibration arthrography in the diagnosis of knee joint diseases]. / Vibrationsarthrographie in der Diagnostik von Kniegelenkskrankheiten.

In this paper, the principles of vibration arthrography are briefly described and the analysis of the resulting signals is explained. Using such a system, localisation of signals from within the joint is possible. Finally, the development of a vibration-based arthrographic system is proposed as a safe, relatively inexpensive, and totally non-invasive aid to the diagnosis of internal disease of the knee.

[Current trends in phonoarthrography]. / Moderne Tendenzen in der Phonoarthrographie.

The detection of sounds from the human body for diagnostic purposes has a long history. In the respiratory and circulatory systems such auscultation is a precise science yielding much useful information. The detection and interpretation of joint sounds, however, has been much less successful in its development. In this paper we outline the history of joint auscultation, of phonoarthrography, and of their modern equivalent: vibration arthrography. The relative merits of acoustic and vibration based systems are discussed and these are illustrated with data from simultaneous recordings of several joint phenomena. Finally, we concluded that the future of phonoarthrography as a clinical test is based on its further development using accelerometers rather than microphones as detectors.