Streptomyces sanglieri which colonised and enhanced the growth of Elaeis guineensis Jacq. seedlings was antagonistic to Ganoderma boninense in in vitro studies.

Actinomycete strain AUM 00500 was 99.5 % similar to Streptomyces sanglieri NBRC 100784(T) and was evaluated for antagonistic activity towards Ganoderma boninense, the causative fungus of basal stem rot of oil palm. The strain showed strong antifungal activity towards G. boninense in in vitro and SEM analysis showed various modes of inhibition of the fungus. Ethyl acetate extracts of single culture and inhibition zone of cross-plug culture by HPLC indicated that strain AUM 00500 produced two different antibiotics of the glutarimide group namely cycloheximide and actiphenol. In greenhouse trials, oil palm seed treated with spores of S. sanglieri strain AUM 00500 at 10(9) cfu/ml showed significant (P < 0.05) increase in oil palm seedlings growth when compared to the control. Streptomyces sanglieri strain AUM 00500 successfully colonised the epidermal surface of the roots of treated oil palm seedlings and it was recovered from root fragments plated on starch casein agar.

Rapid identification of cyclic tetrapyrrolic photosensitisers for photodynamic therapy using on-line hyphenated LC-PDA-MS coupled with photo-cytotoxicity assay.

INTRODUCTION: Photodynamic therapy is a treatment modality that involves site-directed generation of cytotoxic reactive oxygen species by light-activated photosensitisers. OBJECTIVE: In order to rapidly identify new photosensitisers from natural extracts, we developed a liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) method to rapidly identify plant extracts that contain photosensitisers, particularly those possessing a cyclic tetrapyrrole structure. METHOD: Six previously isolated compounds (1-6) were identified in bioactive fractions derived from 15 plant extracts on the basis of their chromatographic retention times, UV-visible profiles, accurate mass and fragmentation patterns. RESULTS: Samples containing uncommon photosensitisers were rapidly identified using this method, and subsequent scale-up isolation efforts led to two new compounds (7 and 8) which were confirmed to be active photosensitisers in a photo-cytotoxicity assay. CONCLUSION: This method serves as a useful tool in prioritising samples that may contain new photosensitisers out of a larger group of photo-cytotoxic natural products extracts.

Sugars consumption by 379 11-12-year-old English children in 1990 compared with results in 1980.

The amount of sugars consumed by young adolescents was assessed in 1990 using the same methods as those employed in a similar survey in 1980. The children were the same age (mean 11 years 6 months) and from the same seven schools in both survey. In 1980, 405 children completed the study and 379 in 1990. Information was collected using two 3-day dietary diaries, each child being interviewed by a dietitian upon completion of a diary. The dietitian in this study was calibrated closely with the dietitian who undertook the 1980 study so as to ensure comparable diet evaluation methods. Total sugars consumption was unchanged (117 g/day in 1980, 118 g/day in 1990) but consumption of non-milk extrinsic sugars increased (83 g/day in 1980, 90 g/day in 1990) and milk and intrinsic sugars decreased (34 g/day in 1980, 28 g/day in 1990) between the two surveys. In 1990, non-milk extrinsic sugars contributed 17% to total dietary energy intake, while milk and intrinsic sugars contributed 5%. There was little difference in percent contributions between the sexes, but some social-class trends were apparent. Confectionery provided 33% and soft drinks provided 27% of non-milk extrinsic sugars, these two dietary sources providing 60% of non-milk extrinsic or 46% of total sugars intake. These levels of consumption are considerably higher than those currently recommended in the UK.

Simulation of cell proliferation in mouse oral epithelium, and the action of epidermal growth factor: evidence for a high degree of synchronization of the stem cells.

Computer simulation has been carried out to help to determine the cell-proliferative mechanisms underlying data gathered from a double-labelling experiment on the dorsal tongue of the mouse. Good fits to the data have been obtained by assuming that there is a high degree of synchrony in the stem cells, which have a 24-h cell cycle time, and that daughters of these cells undergo two further divisions, with mean cell cycle times of 48 h, before differentiating. This results in one-seventh of proliferative cells being stem cells, which ties in well with the concept of epidermal proliferative units. There is no need to assume that S-phase duration changes diurnally. The administration of epidermal growth factor seems to increase the degree of synchrony. In such systems, the influx to S-phase and the efflux from it have very sudden short peaks, which it is impossible to observe unless observations are taken very frequently. There are therefore implications for the designs of experiments that attempt to study diurnal rhythms or the effect of factors that disturb the normal proliferative pattern of cells.

In vitro labelling studies and the measurement of epithelial cell proliferative activity in the human oral cavity.

Samples (110) of human mandibular gingiva and buccal mucosa, harvested from patients undergoing third-molar surgery, were subjected to in vitro labelling with tritiated thymidine, bromodeoxyuridine, or a sequential double-labelling technique comprising tritiated thymidine followed by bromodeoxyuridine, in order to determine the efficacy of a new incubation labelling technique, and to characterize S-phase labelling indices in human oral mucosa. Whilst, there were no demonstrable differences in labelling indices obtained by single thymidine, single bromodeoxyuridine or double labelling, there was a significant difference between anatomical sites, with higher S phase labelling observed in buccal mucosa (mean LI 11.7) than mandibular gingiva (mean LI 8.5; P<0.01). There was, however, no significant correlation between individual labelling indices and patient age, sex or the time of day when tissue was harvested. The in vitro labelling technique provides a reliable and quantifiable method of characterizing proliferative labelling indices in the human oral cavity. Further investigation is being carried out to profile wider age and anatomical ranges and to utilize the double-labelling technique to calculate S-phase durations and cell-cycle times. These profiles may have a future role in the assessment of oral mucous membrane disease.

Using an antedependence test to analyse post-operative pain measurements.

Post-operative pain is frequently measured by a visual analogue scale (VAS). The most common methods for analysing such data are the so-called time-by-time method and repeated measures analysis of variance. In the first, due to correlation between time points, successive tests are not independent and cannot measure the treatment effects over time. In the second, a rather unlikely covariance structure has to be assumed. In a simulation study we have shown that the power of the antedependence test is nearly the same as that of repeated measures of analysis of variance and it does not suffer from some of the latter's shortcomings. We also present an analysis of real data using the antedependence test.

Mapping dynamic epithelial cell proliferative activity within the oral cavity of man: a new insight into carcinogenesis?

Our aim was to characterize epithelial cell proliferative activity within the oral cavity and to find out if there were differences between sites with high and low incidence of cancer. A total of 105 samples of clinically normal mucosa were harvested from various intra-oral sites. Excised specimens were incubated in vitro with tritiated thymidine and bromodeoxyuridine to 'double label' cells undergoing DNA synthesis, and enable calculation of the duration of S phase and estimation of variables of cell flux to and from S. Mean labelling indices (percentage of cells within the S phase of the cell cycle) were highest in the floor of mouth (12.3%) and ventral tongue (10.1%), while activity was lowest in the dorsum of tongue (4.3%) and the palate (7.2%), P<0.001. In general, both cell influx and the duration of S increased proportionally to the labelling index. Sites with a high incidence of cancer were characterized by high labelling indices, increased cell influx and a prolonged S phase.

Characterization of epithelial cell activity in patients with oral cancer.

Accurate, predictive assessment of the clinical behaviour and progression of individual oral cancers and premalignant lesions requires reproducible and quantitative analyses of diseased tissue. In this paper we describe the use of in vitro double labelling (sequential tritiated thymidine and bromodeoxyuridine staining of proliferating epithelial cells) to calculate S phase labelling indices (LIs), estimation of S phase duration (tS), and measurement of variables of flux to and from S for excised specimens of oral squamous cell carcinoma, premalignant lesions, and clinically normal mucosa from patients with oral cancer. There was a significant increase in mean LIs in buccal mucosa leukoplakias (14.5%) compared with normal mucosa (10.3%); P = 0.03. LIs were also increased in patients with cancers of the floor of mouth and ventral tongue but neither these changes nor alterations in flux parameters or S Phase durations were significant. Twenty-one kinetic profiles of dysplastic and malignant tissue were compared with conventional histopathological results, however, and these showed a 2.2% increase in LIs with each increase in grade of dysplasia (P = 0.004) and a 12% increase in LIs with each reduction in tumour differentiation (P = 0.02).

Tritiated thymidine and bromodeoxyuridine double-labelling studies on growth factors and oral epithelial proliferation in the mouse.

Mouse tongue epithelium is characterized by a circadian variation in the number of cells undergoing DNA synthesis. Groups of male BDF1 mice were followed over 48 h and a double-labelling method with tritiated thymidine and bromodeoxyuridine used to determine S-phase labelling indices, together with cell influx to and cell efflux from S, at 4-hourly time points. Control animals exhibited diurnal peaks in labelling index at 03:00 with trough activity 12 h later at 15:00. Cell influx peaked at 23:00 with troughs occurring between 11:00 to 15:00. Peak cell efflux occurred at 07:00 with trough activity at 19:00. Animals injected with epidermal growth factor at 05:00 demonstrated a significant fall in both influx and efflux throughout the 48-h period (P < 0.001), but with preservation of labelling indices, suggesting a slower transit of cells through S-phase, whereas epidermal growth factor injected at 15:00 only produced a significant rise in cell-efflux values. Adrenergic stimulation by intravenous phenylephrine/isoprenaline injection at both 05:00 and 15:00 resulted in a significant rise in cell efflux (P < 0.001), although there was also a rise in labelling index in the 15:00 group (P < 0.001). Animals injected with calmodulin at 05:00 demonstrated a significant reduction in labelling index throughout the 48-h period (P < 0.001), but maintained control values for cell influx and efflux, suggesting faster transit of cells through S. Calmodulin injection at 15:00 produced only a significant reduction in cell influx (P < 0.001). Administration of exogenous growth factors significantly alters the normal rhythmical proliferation of oral epithelial cells in a mouse model. These effects appear to be both growth factor- and time-dependent, and may have both physiological and pathological implications.

Colonic epithelial cell proliferation in hereditary non-polyposis colorectal cancer.

BACKGROUND: Despite the recent discovery of four genes responsible for up to 90% of all cases of hereditary non-polyposis colorectal cancer (HNPCC), there will still be families in whom predictive testing is not possible. A phenotypic biomarker would therefore be useful. An upwards shift of the proliferative compartment in colonic crypts is reported to be one of the earliest changes in premalignant mucosa. AIMS: To assess the role of crypt cell proliferation as a phenotypic biomarker in HNPCC. PATIENTS: Thirty five patients at 50% risk of carrying the HNPCC gene (21 of whom subsequently underwent predictive testing and hence gene carrier status was known) and 18 controls. METHODS: Crypt cell proliferation was measured at five sites in the colon using two different techniques. Labelling index was determined using the monoclonal antibody MIB1 and whole crypt mitotic index was measured using the microdissection and crypt squash technique. The distribution of proliferating cells within the crypts was also assessed. RESULTS: There were no significant differences in the total labelling index or mean number of mitoses per crypt, nor in the distribution of proliferating cells within the crypt, between the study and control groups at any site. When the 21 patients in whom gene carrier status was known were analysed separately there were no significant differences in the measured indices of proliferation between the HNPCC gene carriers and non-gene carriers. CONCLUSION: Crypt cell proliferation is not a discriminative marker of gene carriage in HNPCC.

Sulindac enhances cell proliferation in DMH-treated mouse colonic mucosa.

In a previous study we reported that the NSAID sulindac had a marked inhibitory effect on the development of colonic tumours in mice treated with the carcinogen 1,2-dimethylhydrazine (DMH). In this study we examined the effects of sulindac in respect of cell-kinetic changes in mouse colonic mucosa as determined by flash labelling with the thymidine analogue bromodeoxyuridine (BrdUrd) at varying intervals during the process of colonic carcinogenesis. We also investigated the possibility that these changes may be modulated by misoprostol a prostaglandin E1 analogue. Four groups of 36 mice each were treated for 18 weeks with the following drug/s respectively: (1) DMH; (2) DMH and sulindac; (3) DMH, sulindac and misoprostol; and (4) DMH and misoprostol. Three animals from each group were killed each week between the sixth week and the eighteenth week after the start of the experiment. A 1-h flash label technique was employed and paraffin sections of colonic mucosa were examined. For each animal a total of 50 perfect axially cut crypts were chosen and the following parameters determined: crypt length, labelling index and labelling index distribution: the data were analysed using the computer program GLIM. For each of the four groups, crypt lengths increased significantly with the duration of treatment with no significant difference between the groups. In sulindac-treated animals the labelling index for all positions increased with duration of treatment whereas for animals not treated with sulindac there was no significant difference in labelling index with respect to duration of treatment. The administration of misoprostol did not appear to significantly alter the effects of sulindac. It is postulated that the observed increase in cell proliferation could be a compensatory phenomenon occurring secondary to loss of crypt epithelial cells by apoptosis induced by sulindac. Also the finding of an increase in labelling index mediated by a chemopreventive agent indirectly questions the rationale behind the therapeutic manipulation of crypt cell proliferation in order to reduce the risk of colon cancer.

A survey of croquet injuries.

Of 214 croquet players who responded to a questionnaire, 76 reported at least one injury to hand, wrist or forearm caused by striking the ball. There was no obvious relation to which of the three main grips the player applied to the mallet. Injuries were somewhat more frequent when the mallet shaft consisted of fibreglass than when it was wood, metal or carbon fibre, but a causal relation has not been established. Back injuries seem less troublesome in croquet than in golf.

An organ-culture method for human colorectal mucosa using serum-free medium.

This study describes an explant organ-culture system in which human colonic mucosa can be maintained for prolonged periods in serum-free medium. Following an initial phase of epithelial cell loss, there was intense regenerative activity, with the reformation of tubular crypts. Estimation of crypt lengths revealed a marked reduction after 5 and 9 days in culture with corresponding increases in labelling indices for the whole crypt. The shapes of bromodeoxyuridine (BrdU)-flash-labelling distribution curves were consistent with a proliferative compartment situated within the lower two-thirds of the crypt. We conclude that this is a useful in vitro model for the study of the effects of growth factors and growth-inhibitory agents in respect of cell proliferation in human colonic mucosa.

Detecting poor design, erroneous analysis and misinterpretation of studies.

To detect bad research in published articles, it is necessary to know what to look for. Some examples of the types of poor study design, inappropriate analyses and pitfalls of interpretation which can occur are presented.

What do we mean by a statistical model?

Statisticians are too often satisfied by fitting data rather than investigating the process out of which the data arose. The assumptions on which they base their models may be quite unrealistic, and while it is true that a model should not be more complicated than necessary, neither should it be too simple. Ways of approaching several sets of data from different areas of clinical medicine are considered, and different attitudes to the purpose of modelling highlighted. The transition from smoothing data, through fitting curves, to modelling underlying processes is discussed.

An application of the truncated Poisson distribution to immunogold assay.

An example of the use of the truncated Poisson distribution in an immunogold assay of dystrophin, a gene product of importance in the study of muscular dystrophies, is presented. The practical benefit of using minimum variance unbiased estimators of relevant functions of the parameter of the distribution is considered.