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1.  A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling 
Abstract
In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft™ carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft™ carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the ‘osteoinductive program’. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.
doi:10.1111/j.1582-4934.2009.00807.x
PMCID: PMC3829044  PMID: 19538476
bone morphogenetic protein; Wnt signalling; osteoinduction; calcium phosphate; periosteum-derived cell; tissue engineering; mechanism of action
2.  Characterization of β-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for β1-, β2- and β3-adrenoceptors in rat ileum 
British Journal of Pharmacology  1999;127(4):949-961.
Functional and molecular approaches were used to characterize the β-AR subtypes mediating relaxation of rat ileal smooth muscle.In functional studies, (−)-isoprenaline relaxation was unchanged by CGP20712A (β1-AR antagonist) or ICI118551 (β2-AR antagonist) but shifted by propranolol (pKB=6.69). (±)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (−)-isoprenaline relaxation.BRL37344 (β3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (±)-cyanopindolol, tertatolol and alprenolol. CL316243 (β3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (β3-AR antagonist; pA2=7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that β3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway.The β1-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the β2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551.In rat ileum, β1-, β2- and β3-AR mRNA was detected. Comparison of tissues showed that β3-AR mRNA expression was greatest in WAT>colon=ileum>cerebral cortex>soleus; β1-AR mRNA was most abundant in cerebral cortex>WAT>ileum=colon>soleus; β2-AR mRNA was expressed in soleus>WAT>ileum=colon>cerebral cortex.These results show that β3-ARs are the predominant β-AR subtype mediating rat ileal relaxation while β1-ARs may produce a small relaxation. The β2-AR agonist zinterol produces relaxation through β3-ARs and there was no evidence for the involvement of β2-ARs in relaxation despite the detection of β2-AR mRNA.
doi:10.1038/sj.bjp.0702605
PMCID: PMC1566085  PMID: 10433503
β-adrenoceptors; β3-adrenoceptors; gastrointestinal smooth muscle; relaxation; rat ileum; messenger RNA
3.  Functional and molecular evidence for β1-, β2- and β3-adrenoceptors in human colon 
British Journal of Pharmacology  1997;120(8):1527-1535.
Relaxation of carbachol pre-contracted human colonic muscle to (–)-isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the β-adrenoceptor subtypes involved. β1-, β2- and β3-Adrenoceptor mRNAs were also measured in colonic muscle and mucosa.(–)-Isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50=7.39±0.12, and this response was inhibited by both propranolol (0.1 μM, pKB 8.55±0.12) and the selective β1-antagonist, CGP 20712A (0.1 μM, pKB 8.80±0.20), while the selective β2-antagonist, ICI 118551 (0.1 μM) failed to inhibit isoprenaline relaxation consistently.(–)-Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70±0.17. Propranolol (0.1 μM), CGP 20712A (0.1 μM) and ICI 118551 (0.1 μM) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre-contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized.β1- and β2-Adrenoceptor mRNAs were present in circular/longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. β3-mRNA was also present in both muscle preparations but was not detected in human colonic mucosa.In summary, β1-adrenoceptors are the predominant subtype mediating isoprenaline-induced relaxation of the thin longitudinal smooth muscle of human colon, while β3-receptors do not appear to be involved in these responses. However, β3-adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. β3-Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.
doi:10.1038/sj.bjp.0701056
PMCID: PMC1564619  PMID: 9113375
β3-Adrenoceptors; messenger RNA; human colon; smooth muscle; mucosa
4.  Prescribing behaviour in general practice: the impact of promoting therapeutically equivalent cheaper medicines. 
BACKGROUND: The volume and cost of prescribing varies considerably between practices. This variation is at least in part due to the prescribing behaviour of individual doctors, who are often faced with a range of therapeutically equivalent generic and brand-name drugs. AIM: To assess the impact on general practitioners' prescribing behaviour of promoting therapeutically equivalent lower cost prescribing in conjunction with an incentive scheme. METHOD: Annual prescribing data from before (1992-93) and after (1993-94) implementation of the incentive scheme were compared retrospectively for general practices in the former Northern Regional Health Authority. Main outcome measures were the practices' 1993-94 rates of prescribing relative to those in 1992-93 for 18 drugs prescribed by brand name, of which 10 were targeted in the promotion, and for 14 drugs or classes of drugs either with equivalent cheaper alternatives or of limited clinical value (10 targeted and four not). RESULTS: For 17 of the 18 drugs, brand name prescribing rates were significantly lower in 1993-94. Reductions in rates were greater for the 10 drugs appearing in the scheme's promotional literature. For other cost-saving measures, total prescribing rates were lower for seven classes of drugs, unchanged for one, but higher for the other six, all of which had been targeted. According to the growth in their overall per capita prescribing costs between the two study years, the 499 practices were categorized as low, average or high. Overall costs and individual prescribing rates for the majority of drugs studied were similar for these three practice groups in 1992-93. In 1993-94, practices' changes in prescribing volume differed between the groups, with the lowest increases in the low cost-growth group for all but one of the 32 classes of drugs. CONCLUSION: Generic substitution was more easily implemented than more complex hints regarding cost-saving substitutions. Practices with smaller overall cost growth were making greater use of cost-beneficial prescribing strategies, whether promoted or otherwise. Simple messages may improve the cost-effectiveness of prescribing in the UK. With information support and encouragement, many prescribers appear to have modified their prescribing habits.
PMCID: PMC1312867  PMID: 9115786
6.  Setting standards of prescribing performance in primary care: use of a consensus group of general practitioners and application of standards to practices in the north of England. 
BACKGROUND: There is considerable variation in prescribing, and existing standards against which primary care prescribing is routinely judged consist largely of local or national averages. There is thus a need for more sophisticated standards, which must be widely applicable and have credibility among the general practice profession. AIM: A study aimed to develop a range of criteria of prescribing quality, to set standards of performance for these criteria, and apply these standards to practices. METHOD: A consensus group consisting of eight general practitioners and a resource team was convened to develop and define criteria and set standards of prescribing performance using prescribing analyses and cost (PACT) data. The standards were applied to 1992-93 prescribing data from all 518 practices in the former Northern Regional Health Authority. RESULTS: The group developed criteria and set numeric standards for 13 aspects of prescribing performance in four areas: generic prescribing, prescribing within specific therapeutic groups, drugs of limited clinical value and standards based on prescribing volume. Except for generic prescribing, standards for individual criteria were achieved by between 9% and 34% of practices. For each criterion, a score was allocated based on whether the standard was achieved or not. Total scores showed considerable variation between practices. The distribution of scores was similar between fundholding and non-fundholding practices, and also between dispensing and non-dispensing practices. CONCLUSION: Using a consensus group of general practitioners it is possible to agree criteria and standards of prescribing performance. This novel approach offers a professionally driven method for assessing the quality of prescribing in primary care.
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PMCID: PMC1239506  PMID: 8745847
8.  The use of nitrates, calcium channel blockers and ACE inhibitors in primary care in the Northern Region: a pharmacoepidemiological study. 
1. Prescribing rates for cardiovascular drugs have substantial local variation. The objectives of this study were to determine the prescribing prevalence of nitrates, calcium channel blockers and angiotensin-converting enzyme inhibitors in general practice, to examine the indications recorded for these prescriptions, and to identify which therapeutic areas contribute to the variation in prescribing. 2. Anonymised patient-specific prescription data were taken from computerised records in 41 VAMP research practices in the Northern Region (total population 330,749). All patients who received any prescription for calcium channel blockers, nitrates or angiotensin-converting enzyme (ACE) inhibitors during a 12 month period were included. Prescribing rates were determined in terms of patients per 1,000 population within age, sex and diagnostic groups. 3. Overall, 4.3% of the study population were prescribed one or more of the drugs. There was virtually no prescribing for patients under the age of 35 years, but thereafter the prevalences rose steeply to peak at ages 65-74 years for calcium channel blockers (91 per 1,000 population) and ACE inhibitors (34 per 1,000), and at ages 75-84 years for nitrates (100 per 1,000). Prescribing prevalence amongst the over 85's was less than half the peak rate for each drug group. Rates for men and women were comparable, except for nitrates where men had higher rates. 4. Recorded indication rates for patients with ischaemic heart disease and treated with any of these drugs reached 112 per 1,000 population in the 75-84 age group, and were higher in men than women, at all ages. Hypertension indication rates were substantially higher in women over 65; across the genders the peak rate was 88 per 1,000 for those aged 65-74 years.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1364911  PMID: 7888286
9.  Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins. 
Journal of Virology  1993;67(9):5279-5288.
Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.
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PMCID: PMC237926  PMID: 8102410
10.  Age, sex, and temporary resident originated prescribing units (ASTRO-PUs): new weightings for analysing prescribing of general practices in England. 
BMJ : British Medical Journal  1993;307(6902):485-488.
OBJECTIVE--To derive demographic weightings to replace the existing system of prescribing units used in analysing prescribing by general practitioners in England. DESIGN--The prescribing data for one year from a sample of 90 practices in 80 family health service authority areas were used to calculate the relative frequency with which items were prescribed, for each sex, in nine age bands and for temporary residents. Data on the variation in cost per item by age and sex then allowed estimates to be made of the relative costs for these groups. Integer values for both the item based and cost based weightings were obtained by conversion to optimal integer scales. MAIN OUTCOME MEASURES--Item based and cost based weightings for each of the 18 age-sex groups and for temporary residents. The cost based weightings were considered more appropriate to the context in which the new system was to be used. RESULTS--Prescribing costs increased noticeably, for both sexes, in the middle years (ages 35-64). Compared with the existing system, the cost based weightings (ASTRO-PUs) gave greater weight to patients aged 45 and over, especially those in the 55-64 age band, at the expense of younger patients. Children under 5 received twice as many items as those aged 5-14, but the inexpensiveness of their drugs made the cost based weightings of the two groups equal. Similarly, women were generally given more items than men, but at a lower average cost per item, which reduced differences between the sexes in the cost based weightings. Costs for patients aged 75 and over, compared with those aged 65-74, were higher only for women. CONCLUSIONS--The cost based weightings proposed are believed to reflect the present distribution of prescribing costs, in relation to age and sex, in English general practice. They are intended for use in analyses at practice level.
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PMCID: PMC1678776  PMID: 8305014
11.  Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity. 
Journal of Virology  1992;66(11):6616-6625.
Human immunodeficiency virus type 1 contains a transmembrane glycoprotein with an unusually long cytoplasmic domain. To determine the role of this domain in virus replication, a series of single nucleotide changes that result in the insertion of premature termination codons throughout the cytoplasmic domain has been constructed. These mutations delete from 6 to 192 amino acids from the carboxy terminus of gp41 and do not affect the amino acid sequence of the regulatory proteins encoded by rev and tat. The effects of these mutations on glycoprotein biosynthesis and function as well as on virus infectivity have been examined in the context of a glycoprotein expression vector and the viral genome. All of the mutant glycoproteins were synthesized, processed, and transported to the cell surface in a manner similar to that of the wild-type glycoprotein. With the exception of mutants that remove the membrane anchor domain, all of the mutant glycoproteins retained the ability to cause fusion of CD4-bearing cells. However, deletion of more than 19 amino acids from the C terminus of gp41 blocked the ability of mutant virions to infect cells. This defect in virus infectivity appeared to be due at least in part to a failure of the virus to efficiently incorporate the truncated glycoprotein. Similar data were obtained for mutations in two different env genes and two different target cell lines. These results indicate that the cytoplasmic domain of gp41 plays a critical role during virus assembly and entry in the life cycle of human immunodeficiency virus type 1.
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PMCID: PMC240157  PMID: 1357190
12.  Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity. 
Journal of Virology  1992;66(8):4748-4756.
Many retroviruses, including the human and simian immunodeficiency viruses, contain a leucine zipper-like repeat in a highly conserved region of the external domain of the transmembrane (TM) glycoprotein. This region has been postulated to play a role in stabilizing the oligomeric form of these molecules. To determine what role this region might play in envelope structure and function, several mutations were engineered into the middle isoleucine of the leucine zipper-like repeat of the human immunodeficiency virus type 1 (HIV-1) TM protein. A phenotypic analysis of these mutants demonstrated that conservative mutations (Ile to Val or Leu) did not block the ability of the viral glycoprotein to mediate cell-cell fusion or affect virus infectivity. In contrast, each of the other mutations, except for the Ile-to-Ala change, completely inhibited the ability of the glycoprotein to fuse HeLa-T4 cells and of mutant virions to infect H9 cells. The alanine mutation produced an intermediate phenotype in which both cell fusion and infectivity were significantly reduced. Thus, the biological activity of the glycoprotein titrates with the hydrophobicity of the residue in this position. None of the mutations affected the synthesis, oligomer formation, transport, or processing of the HIV glycoprotein complex. Although these results do not rule out a role for the leucine zipper region in glycoprotein oligomerization, they clearly point to a critical role for it in a post-CD4 binding step in HIV membrane fusion and virus entry.
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PMCID: PMC241301  PMID: 1629954
13.  Accuracy of weighed dietary records. 
BMJ : British Medical Journal  1990;300(6736):1398.
PMCID: PMC1662976  PMID: 2372591
14.  Psychological and social evaluation in cases of deliberate self-poisoning seen in an accident department. 
The outcome in 115 consecutive patients with mild self-poisoning seen by junior medical staff and discharged from the accident department was compared with that of 98 similar patients admitted to the medical wards. Psychiatrists saw only four patients in the accident department and 25 admissions. In making their assessments the junior medical staff considered psychosocial factors as well as the patients' physical condition. Most patients recommended for further care, and discharged from the accident department, subsequently received it. Repetition rates were similar in the two groups and there had been no suicides when patients were followed up at one year. It is feasible for junior staff in an accident department to decide whether patients with self-poisoning need admission or may be discharged with or without subsequent referral for psychiatric or social work help.
PMCID: PMC1496033  PMID: 6800509
15.  Pharmacological screening of new chemical entities in human subjects and methods of data handling. 
1 A system is described for screening new chemical entities in normal human subjects, which is designed to search for novel drug actions at an early stage of drug development. 2 In single (subject) blind experiments, doses are increased incrementally from one subject to next, with immediate feed-back of results from each dose. 3 As many physiological variables as possible are recorded, for which a purpose-designed apparatus system was constructed, and connected to a computer. 4 To overcome problems of inter-subject variability, the need for sensitivity, and to identify drug-related effects from the mass of data generated, a novel data management system was developed, and physiological profiles of volunteer subjects were built up to provide control data. 5 Results from a validation experiment in which isoprenaline was infused intravenously at different rates are presented and discussed.
PMCID: PMC1429994  PMID: 7378250
16.  Deaths from ischaemic heart disease and infant mortality in England and Wales. 
Death rates from ischaemic heart disease (IHD) in English and Welsh counties are correlated, in both men and women, with the infant mortality rates of those counties when the individuals whose deaths are considered were young, thus confirming previous findings in Norway. In England and Wales, however, there is an equally good correlation between deaths from IHD and infant mortality patterns up to and including that for the same time period as the IHD deaths. The British data provide no grounds for concluding from these relationships that living conditions during early life per se bear a causal relationship to deaths from IHD.
PMCID: PMC1051954  PMID: 508999
17.  Consultation-liaison scheme for self-poisoned patients in a general hospital. 
British Medical Journal  1978;2(6149):1392-1394.
In a prospective clinical trial 276 self-poisoned patients consecutively admitted to hospital were randomly allocated to medical teams or to psychiatrists for an initial psychiatric assessment and a decision about "disposal." Junior doctors and nurses received instruction in this work. While awaiting the outcome of the trial the randomisation was continued for 13 months and 729 allocations were made altogether. Physicians requested psychiatric opinions for roughly one in five of their patients. In other respects medical teams performed similarly to psychiatrists. Provided that due attention is given to teaching junior staff and to ensuring that psychiatric treatment and social-work support are available once patients have been assessed, such a consultation-liaison scheme could be adopted in other hospitals. This would help to change unfavourable attitudes towards self-poisoned patients and contribute to the general training of doctors and nurses.
PMCID: PMC1608625  PMID: 719418

Results 1-17 (17)