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The results of a ‘BioWise’ demonstration project to assess the comparative sensitivity and practicality of seven new assays for the direct assessment of ecotoxicity in industrial effluents are presented. In addition the aim of the project was to validate the results of the new assays against benchmark data generated from non-proprietary, rapid, microplate screening assays using the regulatory species; freshwater crustacean Daphnia magnaand green algae Selenastrum capricornutum, chosen in view of their environmental relevance. The new commercial test assays were: Daphnia magna, Selenastrum capricornutumand Thamnocephalus platyurusToxkits® supplied by Vickers Laboratories Ltd, containing dormant, immobilised life stages of the test species; GreenScreen® EM, a yeast based assay for genotoxicity and general acute toxicity supplied by Gentronix Ltd; and CellSense® a mediated, amperometric whole cell biosensor based on immobilised activated sludge and E. coli. 38 effluent samples supplied by members of SOCSA Specialised Organic Chemicals Sector Association were examined over a period of 13 months, in the project co-ordinated by the AstraZeneca Brixham Environmental Laboratory, and part funded by BioWise viathe UK Government Department of Trade and Industry.

The results of a ‘BioWise’ demonstration project to assess the comparative sensitivity and practicality of seven new assays for the direct assessment of ecotoxicity in industrial effluents are presented. In addition the aim of the project was to validate the results of the new assays against benchmark data generated from non-proprietary, rapid, microplate screening assays using the regulatory species; freshwater crustacean Daphnia magna and green algae Selenastrum capricornutum, chosen in view of their environmental relevance. The new commercial test assays were: Daphnia magna, Selenastrum capricornutum and Thamnocephalus platyurus Toxkits® supplied by Vickers Laboratories Ltd, containing dormant, immobilised life stages of the test species; GreenScreen® EM, a yeast based assay for genotoxicity and general acute toxicity supplied by Gentronix Ltd; and CellSense® a mediated, amperometric whole cell biosensor based on immobilised activated sludge and E. coli. 38 effluent samples supplied by members of SOCSA (Specialised Organic Chemicals Sector Association) were examined over a period of 13 months, in the project co-ordinated by the AstraZeneca Brixham Environmental Laboratory, and part funded by BioWise via the UK Government Department of Trade and Industry.

The jellyfish green fluorescent protein (GFP) is a versatile biological marker and reporter. Here we demonstrate that marked polarisation in GFP fluorescence, upon excitation with plane polarised light, can be exploited to enhance the detection of GFP when in the presence of cellular auto-fluorescence and other fluorescent compounds that emit at the same wavelength. The development of flow-though instrumentation dedicated to the sensitive detection of GFP by fluorescence polarisation is described, and used in both a continuous flow and flow-injection format. The intensity, spectral properties and extent of polarisation in the auto-fluorescence of various yeast strains and growth media are investigated. The application of fluorescence polarisation is shown to enhance the measurement of the induction of GFP in yeast cells, genetically modified to produce GFP as an indicator of DNA damage, compared with a conventional fluorescence method.

Saccharomyces cerevisiae has been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombe also undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process. Copyright © 1999 John Wiley & Sons, Ltd.

Saccharomyces cerevisiaehas been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombealso undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process. Copyright © 1999 John Wiley & Sons, Ltd.

Recent proposals to analyze a number of major genomes have created a need for methods of rapidly manipulating and assaying very large numbers of specimens containing DNA fragments. Systems for sampling semi-solid biological material from mixed disordered arrays are required. These samples need to be sorted in an ordered format and stored in minimum space at known locations. It should be possible to recall them quickly from stock, individually or collectively, for duplication or re-ordering into new arrays for appropriate analysis. This article reviews a range of problems associated with high speed multiple specimen handling and assay in the molecular biology laboratory and outlines the solutions currently available to address these difficulties. Designs for automatic systems to manipulate bio-samples and analyze DNA are presented.

The green fluorescent protein (GFP) of Aequorea victoriais now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts—a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54gene. It is shown that the induction of the RAD54promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disruptants for their effects on RAD54expression and so identify trans‐acting factors involved in the cellular response to DNA damage. © 1997 John Wiley & Sons, Ltd.

The objectives and rationale of the First International Symposium on Southern Hemisphere Limnology are outlined. The geography and climate of the Northern and Southern Hemispheres are compared. Greater areas of the land masses in the Southern Hemisphere fall under oceanic influence than in the Northern Hemisphere at equivalent latitude. Although many Southern Hemisphere land masses are arid it is concluded that limnological differences between the Hemispheres are of degree rather than of kind and that limnological principles developed in the Northern Hemisphere are, in general, globally applicable. However, Southern Hemisphere water resources management problems have centred upon arid-land water supply problems, probably to the detriment of ‘pure’ limnological research.

Transcutaneous nerve stimulation has become a popular and widely accepted method for controlling patients' pain. Although it is now frequently used in physical therapy departments, the physiological mechanism whereby it brings about its results is poorly understood. It was hypothesised that the application of TNS to naive normally healthy (n= 12) students would produce a reduction in the sensory nerve conduction velocity of the median nerve. This proved to be so (p = .003), but unexpectedly one subject reported a feeling of coolness in the exposed hand. Subsequent analysis of cutaneous skin temperature showed a statistically significant reduction (p < .001). Correlation coefficients showed higher correlations between the temperature-temperature and velocity-velocity changes than those between temperature-velocity. One deduced that the reduction in nerve conduction was therefore primarily the result of the TNS, although further study of this topic is planned.

A 2.2 kb Sphl/Clal fragment of the Clostridium pasteurianum chromosome has previously been cloned and shown to complement leuB401 and leuC171 mutations in Escherichia coli. The nucleotide sequence of this fragment has been determined (2327 bp) and carries three open reading frames. The products of translation of these reading frames display significant homologies with the α-isopropylmalate isomerase subunit (leuD) gene of Salmonella typhimurium, the β-isopropylmalate dehydrogenase (leuB) genes of several organisms, and the dihydroxyacid dehydrase (ilvD) gene of E. coli.

Measurements of plasma apolipoprotein A-1 and B concentrations are increasingly used for the laboratory assessment of risk of coronary artery disease (CAD). This study of 22 patients investigated the response of plasma apolipoprotein A-1 and B levels for up to 20 days following a myocardial infarction. Seven of these patients participated in a clinical trial using the drug Tissue Plasminogen Activator (TPA). We established that, unlike many other plasma proteins, apolipoproteins do not display a classic acute phase response following myocardial infarction, although large variations in plasma apolipoprotein levels were observed in the patients investigated. Our studies also show that the measurement of plasma apolipoproteins A-1 and B to assess future CAD risk in myocardial infarction patients should be deferred for a minimum of at least 14 days post-infarction. No significant difference was observed in the pattern of apolipoprotein response between patients receiving TPA and those not given this drug.

This study was carried out to characterize the antibody class response by ELISA to seven Klebsiella pneumoniae serotypes (K2, K3, K17, K21, K26, K36, K50) in five different groups, 40 HLA-B27-positive ankylosing spondylitis (AS) patients, 46 patients with Crohn's disease (CD), 38 patients with ulcerative colitis (UC), 50 patients with active anti-endomysial antibody-positive coeliac disease and 40 healthy controls, using whole bacteria and capsular polysaccharide. IgG antibody levels were significantly elevated in AS patients to K17, K36, K50; IgA to K2, K3, K21, K26, K36 and K50; and IgM to serotype K21 when compared to normal controls. Furthermore, IgG antibody levels were significantly elevated in CD patients to K2, K17, K21, K26, K36 and K50; IgA to K2, K3 K21, K26, K36 and K50; and IgM to K2, K3, K17, K21 and K50. Increased IgG antibody levels in the UC group were limited only to K17, K35 and K50. No antibody class was increased to any of the K. pneumoniae serotypes other than K26, K36 and K50. The similarity in the immune response between CD and AS groups suggests that many AS patients may have occult bowel inflammation.

Chlorophyll adata from 24 South African impoundments were examined with the objective of developing a trophic status classification system. For the combined data set, standard deviation of the mean annual chlorophyll aconcentration displayed an almost 1:1 linear relationship to the mean, indicating a wide annual variation in chlorophyll afor most impoundments. There appeared to be no common seasonal pattern to the occurrence of chlorophyll amaxima and minima, leading to the conclusion that trophic status was best described by a statistical parameter based on annual data (notably the mean value). Statistically significant empirical formulae that relate both the maximum value and the frequency of occurrence of specific chlorophyll aranges to the mean annual value were established, and their potential use in the assessment of trophic status and impoundment management is described.

An investigation of the phosphorus loading characteristics of 31 southern African man-made was lakes made. The lakes were characterized by low water retention times, with most of the lakes having retention times of less than one year. Catchment phosphorus export rates showed wide variation (1–162 mg P m-2 y-1) with those lakes experiencing excessive municipal wastewater inputs having export rates in excess of 53 mg m-2 y-1. The phosphorus data were tested against the Vollenweider (1976) and Dillon & Rigler (1974) phosphorus budget models which predict in-lake steady state concentrations of phosphorus. It was found that both models displayed good potential for the prediction of steady state concentrations of phosphorus, with better results being obtained from the Dillon & Rigler (1974) model. However, because phosphorus concentrations within these lakes may not necessarily be related to trophic status the use of these models as a predictive tool for eutrophication control still requires further development.

An investigation of water transparency characteristics and light attenuation by waters of a turbid subtropical impoundment showed that allochthonous inputs of silt during summer floods, impoundment morphometry, and the warm monomictic thermal cycle were the main factors regulating the temporal and spatial variations in water transparency. Statistically significant relationships between the Secchi disc transparency, turbidity of the surface water, mean diffuse attenuation coefficient and beam attenuation coefficient, were established. These relationships allowed for an approximation of the 1 per cent of surface light intensity depth to be made by using any of the four parameters. The attenuation of blue light was greater than that of red light, owing to the effects of suspended clay particles on the spectral attenuation of light.

Summary Penetration of the phloem of young and mature leaves ofNerium oleander by stylets of the aphid,Aphis nerii was studied with light, phase and differential interference contrast microscopes. Two of five pairs of stylet tips encountered in young leaves and eleven of sixteen pairs encountered in mature leaves were lodged in sieve tubes of the adaxial phloem. In young leaves, the majority of penetrations originated from the abaxial epidermis; conversely, the majority of penetrations in mature leaves originated from the adaxial epidermis. In all instances, penetration of the epidermis, ground tissue and phloem was largely intercellular.

Summary Penetration of the stems and leaves ofGomphocarpus physocarpus (E.Mey) by the aphid,Aphis nerii (B. de F.) was studied with light and phase microscopes. Penetration of the epidermis and ground tissues was largely intercellular, that of the phloem tissues partly intercellular and in part intracellular. In the large majority of penetrations the external phloem was bypassed, the stylet tracks terminating in the sieve tubes of the internal phloem. Of 75 pairs of stylet tips encountered in presumably functional sieve tubes 73 were lodged in sieve tubes of the internal phloem. This confirms observations of a preliminary study which indicated thatA. nerii feeds preferentially on sieve tubes of the internal phloem. A satisfactory explanation of this preferential feeding has yet to be provided.

Alumina-titanium carbide composite sliders used in magnetic recording were coated with diamondlike carbon (DLC) to permit exploration of their effect on tribochemical wear. For comparison, testing was performed on both coated and uncoated sliders. Gases sampled directly from the sliding interface between a carbon-coated thin-film magnetic recording disk and the slider contained carbon dioxide in both dry nitrogen and dry oxygen environments. In the oxygen environment, uncoated sliders produce carbon dioxide at a rate 10 times greater than coated sliders. This suggests that catalysts in the slider composite material are necessary for carbon oxidation wear.

Lubricant loss, degradation and mobility of perfluorinated poly-ether (PFPE) on carbon coated disks were investigated by observing friction during sliding contact while controlling temperature and humidity. Lubricant loss from asperity contacts and its replenishment from adjacent areas were investigated for Al2O3-TiC and Mn-Zn ferrite sliders at various sliding velocities by continuous and oscillating rotation of the disk.

The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts—a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disruptants for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage. © 1997 John Wiley & Sons, Ltd.

BACKGROUND: The appropriate medical treatment of patients with ulcerative colitis is determined largely by the severity of symptoms. Hospital assessment of the severity of disease activity includes investigation of laboratory indices and sigmoidoscopic assessment of mucosal inflammation. AIMS: To develop a simplified clinical colitis activity index to aid in the initial evaluation of exacerbations of colitis. METHODS: The information for development of the simple index was initially evaluated in 63 assessments of disease activity in patients with ulcerative colitis where disease activity was evaluated using the Powell-Tuck Index (which includes symptoms, physical signs, and sigmoidoscopic appearance). The new index was then further evaluated in 113 assessments in a different group of patients, by comparison with a complex index utilising clinical and laboratory data, as well as five haematological and biochemical markers of disease severity. RESULTS: The newly devised Simple Clinical Colitis Activity Index, consisting of scores for five clinical criteria, showed a highly significant correlation with the Powell-Tuck Index (r = 0.959, p &lt; 0.0001) as well as the complex index (r = 0.924, p &lt; 0.0001) and all laboratory markers (p = 0.0003 to p &lt; 0.0001). CONCLUSIONS: This new Simple Colitis Activity Index shows good correlation with existing more complex scoring systems and therefore could be useful in the initial assessment of patients with ulcerative colitis.

The chromosomes of the yeast Saccharomyces cerevisiae terminate with sequences that have the form poly(C1-3-A). In this paper, we show that within an individual yeast strain all chromosomes end with tracts of poly(C1-3-A) of similar lengths; however, different strains can have tracts that vary in length by a factor of two. By a genetic analysis, we demonstrate that yeast cells have a mechanism that allows them to change rapidly the length of their chromosomes by altering the length of the poly(C1-3-A) tract.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

Abstract: Specific immunoreactive anti-Klebsiella antibodies are found in patients with ankylosing spondylitis (AS), a significant proportion of whom have occult inflammatory bowel disease. Molecular mimicry between Klebsiella or other bacterial antigens and HLA-B27 has been suggested in the pathogenesis of AS. The specificity of increased immunoreactivity against Klebsiella remains to be assessed against the abundant anaerobic bacterial flora, present either in healthy controls or in patients with ulcerative colitis (UC) and Crohn's disease (CD). Total immunoglobulin (Ig; IgG, IgA, IgM) immunoreactivity was measured by ELISA against Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli and ten anaerobic isolates of the predominant normal bowel flora in 35 patients with active AS, 60 patients with inflammatory bowel disease (30 CD, 30 UC), 60 patients with active rheumatoid arthritis (RA) and 60 healthy controls. Ig immunoreactivity to K. pneumoniae was significantly elevated in AS (P < 0.001), CD (P < 0.001) and UC (P < 0.001) patients compared with RA patients and healthy controls. Furthermore, Ig immunoreactivity to P. mirabilis was significantly elevated only in RA patients, compared with the other inflammatory groups (P < 0.001) and controls (P < 0.001). There was no significant antibody response against E. coli or the ten obligate anaerobes in any of the test groups. The data suggested an increased immune response to Klebsiella in patients with AS, UC, CD and to Proteus in patients with RA. The specificity of these responses in some patients supported a possible role for enteric Klebsiella in the pathogenesis of AS and Proteus in RA. The role of Klebsiella in inflammatory bowel disease requires further study.

1. A series of experiments on adult rabbits was carried out in which a tendon was transplanted and embedded in a bony tunnel and traversed a joint after the manner of a tenodesis.2. Histological observations were made on the reaction of surrounding bone and tendon at intervals over a period of 307 days.3. The findings suggest that the buried tendon undergoes a process of progressive degeneration, and that host cells issuing from the adjacent bone marrow infiltrate and ultimately replace it by new tendon tissue.4. The invading cells are believed to be derived, as a result of the provocative stimuli provided by the experiment, from primitive reticular cells of the haemopoietic tissue.5. The tunnelled bone undergoes considerable remodelling and associated with this is the presence of a considerable number of osteoblasts and osteoclasts.

1. The normal anatomy of the intervertebral disc of immature rabbits is described.2. An account is given of the changes that occur after an operative incision in the ventral part of the intervertebral discs of rabbits which allowed the escape of the nucleus pulposus. The account is based on observations made on fifty-five young animals killed at intervals during the twenty-five months after operation.3. The superficial part of the wound in the annulus heals rapidly by active fibrosis. Thereafter there is a chondrification of the ventral region of the disc, followed by ossification. A prominent bony ridge ultimately ankyloses the vertebrae adjoining the disc.4. The site of the nucleus pulposus is eventually occupied by a dense pad of fibrocartilage. A tongue of this tissue projects into the deep median part of the wound which remains unhealed.5. A hypothesis is submitted regarding the mechanism of rupture of the annulus fibrosus and prolapse of the nucleus pulposus in man; this hypothesis is based in part on the observations of lesions in discs not subjected to operation.

The temperature dependence of mating-pair formation by Escherichia coli has been studied from 2 to 45 C by means of a physical assay. No mating-pair formation was found below 24 C. Between 30 and 41 C pair formation increased very rapidly, followed by an equally sharp decline between 41 and 45 C. The results are interpreted in terms of the pilus adsorption process of recipient cells and pili production by donor cells.

Yeast chromosomes terminate in a GC-rich tail of DNA. Previous investigations have shown that the length of this tail can change in response to genetic variation. Here we present data that show that the length can also alter in response to changes in the amount of the GC-rich DNA found elsewhere in the nucleus.

Doctors are optimistic in their perception of how acceptable endoscopy is for patients. We analyzed elements that contribute to a poor experience for the patient and the agreement between the perceptions of endoscopists and patients. Eighty-four outpatients who had undergone gastroscopy completed questionnaires (response rate of 73%) 48 to 96 hours after the procedure. The endoscopist completed a similar questionnaire. Questions concerned overall tolerance, swallowing, retching and vomiting, sedation, duration, diagnosis, age, and sex. Data from both doctor and patient were available in 84 cases. The type of sedation, administration of hyoscine or Xylocaine (Astra Pharmaceuticals Ltd., Kings Langley, UK), diagnosis, and expression of need for more sedation were not statistically significantly related to the overall patient score. The largest contribution to a poor overall tolerance score arose from difficulty in swallowing the endoscope, followed by the duration of the procedure. A total of 8.3% of patients reported some overall difficulty which was not recognized by the endoscopist. In relation to retching and vomiting, 11.9% of patients had difficulty with retching and vomiting, which went unrecognized by the doctor, and 18% had difficulty in swallowing the endoscope. There was fair agreement between the assessments of overall acceptability of both endoscopists and patients. Difficulty in intubation, however, is the major contributor to a poor tolerance of gastroscopy and also the issue on which doctors and patients disagreed the most.

The results of in‐vitro thyroid function tests and clinical details of 1,517 patients are reviewed, and the total serum thyroxine levels and the free thyroxine indices are compared in terms of their diagnostic value. Only 3.7% of the patients investigated had a total serum thyroxine level (T4) that did not correlate with the free thyroxine index. We believe that the evidence is strong enough to suggest that estimation of the T4 is an adequate screening test for thyrometabolic disease, and that performing the extra test (T3 resin uptake) in order to estimate the free thyroxine index (FIT) cannot be justified in terms of cost‐benefit. We suggest that the FTI is indicated only in those cases in which the T4 is outside the 80% limit range. A simple scheme for the investigation of thyrometabolic disorders is presented.