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.

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.

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.

The proliferative response of the coagulating gland of the castrated mouse under continuous androgen stimulation: an experimental and computer simulation model.

The proliferative response of the coagulating gland of the castrated male mouse has been examined during continuous treatment with testosterone propionate. Fourteen days after castration, s.c. daily injections of testosterone propionate were begun. Mitotic (Im) and labelling (IL) index values were obtained at 3 h intervals for up to 100 h after the initial injection. These showed a biphasic response, in which IL reached a maximum at 30 and 70 h, and Im at approximately 45 and 75 h. Fraction-labelled mitoses (FLM) curves were begun 24, 48, and 72 h after the first androgen injection. In each curve the first wave of labelled mitoses rose to 100% and showed a square form indicating little spread in the durations of the G2 and S phases. Values of 7.5, 1.3 and 0.7 h were obtained for the durations of DNA synthesis (ts), the post-synthetic period (tG2) and of mitosis (tm) respectively. In none of the FLM curves was it possible to demonstrate a second wave of labelled mitoses and direct measurement of the cell cycle time (Tc) was not obtained. Continuous tritiated thymidine labelling indices revealed that after a latent period of 25 h, DNA synthesis began and labelling rose rapidly to 80% by 45 h and then more slowly to 95% by 97 h. Cell population changes during androgen stimulation estimated from measurements of total glandular DNA indicated that the number of cells present in the glands remained constant during the first 30 h after stimulation and thereafter increased to approximately 2-3 times the original value. The data are compared with a mathematical model which assumes that the cell population of castrated mice when stimulated passes from a GO compartment through successive waves of DNA synthesis and mitosis. After each cell division the cells may leave or remain in the proliferative cycle. This model has been subjected to computer simulation using the cell cycle parameters obtained in the kinetic experiments. There was good agreement between the stimulation and experimental results in the Im and IL curves, continuous labelling, and total cell number experiments. The simulation of FLM curves was less successful. Although the first wave of labelled mitoses was clearly seen the model predicts a distinct second wave of labelled mitoses. It is concluded that this does not appear because of variation in the duration of G1.

Modulation by verapamil of vincristine pharmacokinetics and sensitivity to metaphase arrest of the normal rat colon in organ culture.

The in-vitro pharmacokinetics of vincristine (VCR) in normal rat colonic mucosa were studied. Two complementary approaches were adopted using an explant organ-culture system. Firstly [G-3H]vincristine (3HVCR) accumulation, retention and efflux were characterized under basal conditions and compared with measurements made either under energy-depleted conditions, or in the presence of VRP. Secondly, a histological method--the postmetaphase index (PMI)--was used to compare the sensitivity of explants to VCR in the presence or absence of verapamil (VRP). This latter technique involves the measurement, by counting, of the proportion of mitotic figures escaping from metaphase arrest. The studies yielded the following results: 3HVCR accumulation in colonic mucosa showed no evidence of saturability up to the maximum dose studied (130 nM), at a dose of 52 nM accumulation was enhanced in energy-depleted conditions by a factor of 1.8, and in the presence of VRP (6.6 microM) by a factor of 1.4. In the presence of VRP (6.6 microM) retention of 3HVCR was increased by a factor of 1.3 and efflux was reduced by a factor of 0.8 after 2 hr. VRP (6.6 microM) reduced the PMI of colonic mucosal epithelial cells exposed to 11 nM VCR from 18.8% to 11.4% (i.e. 40% reduction) indicating sensitization of the cells to this property of VCR. These results provide evidence that the sensitivity of normal colonic mucosa to vincristine is, at least in part, regulated by drug transport. Qualitatively our observations resemble those described in multidrug resistance. Given that P-glycoprotein has been demonstrated by several groups in colonic mucosal cells, the results support a normal role for this membrane transport molecule in the protection of intestinal cells from plant alkaloids and other xenobiotic agents ingested in the diet.

Cytokinetic studies of crypts in convoluted human small-intestinal mucosa.

Forty-seven peroral biopsy specimens of duodenojejunal mucosa showing convolutions were obtained from patients with a variety of clinical disorders. These mucosal samples were divided into three groups according to the extent of the convolutions and the severity of the accompanying histopathological changes; the cytokinetic status of the crypts in the three groups was then analysed and compared. Those in which the mucosae were predominantly or totally convoluted (group 3) showed the most notable cytokinetic changes: crypts were hyperplastic and crypt cell production rate was markedly increased compared with the other two groups and with morphologically normal control mucosae. In the case of one patient with mucosal changes of group 3 severity, additional studies were carried out using vincristine to produce metaphase arrest. The cell cycle time of 27 hours was greatly shortened compared with a control value of 45 hours.We find that the presence of convolutions in small-intestinal mucosal biopsy specimens is accompanied by an increase in the rate of cell production from the crypts, which is presumably related directly or indirectly to the rate of loss of mature enterocytes from the surface of the mucosa. Furthermore, an increase in the proportion of convolutions may reflect an increase in the rates of cell production and cell loss. In the group 3 convoluted mucosae the cytokinetic profile of the crypts resembled that of some of the flat avillous coeliac mucosae previously studied although the rates of cell production did not reach the levels attained by the most productive of the flat coeliac mucosae.

Presence of epidermal growth factor receptor as an indicator of poor prognosis in patients with breast cancer.

Epidermal growth factor receptors are present in some breast cancers in man, and there is an inverse relation to oestrogen receptor state. We assessed the presence of epidermal growth factor receptors as a single prognostic indicator in a series of breast tumours by comparing this with the Bloom and Richardson scores for these tumours. One hundred and eight ductal tumours were examined for epidermal growth factor receptors by radioligand binding. There was a significant (p less than 0.01) correlation between the presence of the growth factor receptor and poor prognosis as assessed by the Bloom and Richardson score, suggesting that epidermal growth factor receptor state could be a useful prognostic marker. Epidermal growth factor receptor state was not significantly correlated with the lymph node state but showed a tendency to be associated with large tumours.

Interpreting the correlation coefficient when one of the variables is discrete.

The effect on the correlation coefficient of discretizing data was investigated in two ways. First, the theoretical effect of dichotomizing data was calculated, and it was shown that the resulting correlation coefficient is considerably less than that between the underlying bivariate normally distributed variables. Second, computer simulations were performed of a model in which a continuous variable (measured with some error) gives rise to a counting variable through a mechanism in which the count is zero below a certain threshold value for the continuous variable and then increases linearly as the continuous variable increases. It was shown that the correlation coefficient between the observed values of the continuous and counting variables decreased as (a) the measurement error increased, (b) the slope of the relationship decreased, and (c) the number of counts decreased. It is concluded that caution is required when interpreting correlation coefficients when one or both of the variables consist of a few (say only four or five) discrete scores.

The distribution of the number of maxima in a cyclic sequence.

A simple statistical method is presented which tests for non-randomness in a cyclic sequence. The distribution of the statistic is tabulated for short sequences, and a large sample approximation is derived. Significant values of the statistic are given for sequences of length up to 52. Easily used computer programs to carry out appropriate calculations are presented.

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.

Relationship between dietary habits and caries increment assessed over two years in 405 English adolescent school children.

The study was conducted in seven schools on children initially aged 11.5 years. They recorded their diet on five occasions, each of three days, and received an annual dental examination, including radiography. Caries increments were low, mostly (58 per cent) in fissure surfaces. Correlations between caries increment and dietary factors were low due to the low caries increments observed and the large error associated with dietary data where analyses attempt to discriminate between individuals. The highest correlation was between caries increment and weight of daily intake of sugars (+0.143, p less than 0.01). Multivariate analyses revealed that this relationship could not be explained by differences in sex, social class, tooth-brushing habits or level of plaque as measured by gingival inflammation. Weight of sugar intake appeared to be more strongly correlated to caries than frequency of intake; concentration of sugars in foods was positively related, and sugars in snacks were more strongly related to caries than total dietary sugars. The 31 children who consumed most sugar (greater than 163 g/day) developed 5.0 DMFS during the 2 years, 0.9 DMFS per year more than the 31 children (3.2 DMFS during 2 years) who had the lowest sugar intake (less than 78 g/day).

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.

Particle release from haemodialysers.

Release of particles from eight makes of hollow-fibre and one make of flatplate dialyser have been studied, and the relationship between rinsing volume and particles recovered established. In a supplementary study the effect on the number of particles released was assessed when striking the dialyser header during priming. Particle size distribution indicated that the majority of the particles recovered were less than 5 microns in diameter. A separate analysis of the particles in the 5-30 micron size range showed two different patterns of particle release. In the dialysers containing ethylene vinyl acetate (EVAL) or Cellulate the number of particles recovered were within the 95% range of the particles in the rinsing fluid and did not alter with increasing rinsing volume. In the dialysers containing Cuprophan and Hemophan the initial particle numbers were higher but fell rapidly up to a 450 ml rinse volume; increasing the rinse volume to 1050 ml did not alter particle recovery. Analysis of variance failed to differentiate between individual dialysers. Striking the header during priming resulted in a transient statistically nonsignificant increase in the number of particles recovered.

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.

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.

Sugars consumption of Northumbrian children aged 11-14 years.

In order to investigate the relationships between diet and tooth decay the authors recently completed the first longitudinal study of diet and dental caries increment in children. 405 children initially aged 11.5 years each recorded their intake of foods and drinks for a total of 15 days over a period of 2 years. This is a review of some of the findings.

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).

Adaptive cell-proliferative changes in the small-intestinal mucosa in coeliac disease.

Cell proliferation in the small-intestinal crypts of rodents has been intensively investigated and lends itself to the deployment of techniques which are inapplicable in man. In particular there are ethical and economic objections to methods involving the use of tritiated thymidine in vivo for specific labelling of DNA, while the validity of in vitro studies using organ culture is uncertain. It is possible, nevertheless to construct a profile of the size and cytokinetic status of the mucosal crypts by analysis of serial sections prepared from well orientated routine diagnostic biopsy specimens. Such studies can provide a measure of mean total crypt-cell population, and by studying the distribution of mitoses in the crypts relative measurements can be obtained of proliferation and maturation compartment sizes, and of crypt cell production rate (CCPR). These parameters have been compared in 62 patients with 'flat' avillous coeliac mucosae and in 85 patients with normal villous mucosae. A heterogeneous group of 47 patients with lesser degrees of abnormality (convoluted mucosae) were similarly studied. In addition, estimates of cell cycle times were obtained in a small group of patients with normal, convoluted and 'flat' mucosae by taking biopsies before and after the administration of the metaphase-arresting agent vincristine. 'Flat' coeliac mucosae show a threefold increase in the size of the proliferation compartment compared with normal and the cell cycle time is approximately halved leading to a net sixfold increase in CCPR. This is the basis of the change in mucosal morphology and presumably represents a compensatory reaction to the gluten-induced increase in loss of enterocytes from the mucosal surface. Convoluted mucosae occupy an intermediate position in terms of the parameters studied and should be regarded as stages in a continuum of adaptive change.