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We utilised genetic markers (ISSR's) to investigate population structuring inCrambe maritima, a coastal perennial found in isolated populations on either side of the English Channel. Despite the expectation that smaller populations would contain lower levels of genetic diversity, we found no correlation with total population size or the number of flowering plants and genetic diversity. Although populations were genetically differentiated from each other, isolation by distance was not apparent in the whole data set or within the UK samples.Despite being sampled from a wide geographic range, the majority of diversity (71%) was contained within populations, with no unique genotype, or even allele, identifying any population. When genetic distances were plotted using MDS techniques, samples from each population had a tendency to cluster, but the spread of points was wide indicating high levels of gene flow. High levels of gene flow, through the dispersal of seeds, were verified by assignment testing, which showed that 8.7% of individuals were assigned to a population other than the one they were collected from. The pattern of genetic variation can be explained by examining the direction of currents in the English Channel and Bay of Biscay. Gene flow, via seed dispersal, is possible between UK and French populations on either side of the English Channel. In contrast, French populations on the Bay of Biscay coast are effectively isolated by the direction of currents and consequently show a greater degree of genetic differentiation. [ABSTRACT FROM AUTHOR]

1. Three experiments were conducted with broiler chickens using hypobaric chambers and control pens, feeding diets containing 25 or 50 g flax oil/kg food or control diets with equivalent amounts of animal/vegetable (A/V) blend oil for 4 weeks. The effect of these diets on haematological variables and the extent of right ventricular hypertrophy (RVH) leading to ascites was determined. 2. Overall growth rate was not consistently affected by dietary treatment, although feeding the 25 g flax oil/kg diet reduced weight gain in week 4 of one experiment. Feeding the 50 g flax oil/kg diet but not the 25 g flax oil/kg diet reduced RVH in birds exposed to hypobaric conditions compared to feeding control diets. 3. Feeding the 50 g flax oil/kg diet under hypobaric conditions reduced the haematocrit and haemoglobin content, increased the erythrocyte deformability and the proportion of unsaturated fatty acids in the erythrocyte membranes, and reduced the whole blood viscosity compared to feeding control diets. These effects were not seen when the 25 g flax oil/kg diet was fed. The ratio of n-3 to n-6 fatty acids in erythrocyte membranes was increased in the 50 g flax oil/kg treatment group compared to controls. 4. Including 50 g flax oil/kg broiler diet reduces RVH in broiler chickens. This may be attributable in part to an increase in erythrocyte deformability from an increased proportion of unsaturated fatty acids in the erythrocyte membranes. [ABSTRACT FROM AUTHOR]

The smooth snake, Coronella austriaca, is particularly rare within the UK and little is known about the genetic variability within the species. Here we report the isolation of 16 polymorphic microsatellite markers in the smooth snake, using a simple enrichment procedure. Despite screening individuals from a potentially isolated population, levels of polymorphism were high. These microsatellite markers will prove very useful in investigating population parameters, which will lead to effective conservation and management plans for this rare species. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of secondary motor tasks of three levels of difficulty on the spatial and temporal parameters of gait in subjects with Parkinson disease (PD) compared with control subjects., Design: A two-group repeated measures design using a sample of convenience. Subjects performed 10-meter gait trials walking (1) freely, (2) carrying a tray, and (3) carrying a tray with four plastic glasses., Setting: Subjects were tested in the gait laboratory at Kingston Centre, Victoria, Australia., Subjects: Twelve subjects with PD and 12 control subjects matched for age, height, and sex were recruited from the Movement Disorders Clinic at Kingston Centre., Outcome Measures: Gait speed, stride length, cadence, and the proportion of the walking cycle spent in double limb support were measured in moderately disabled subjects with PD and control subjects., Results: For all of the walking conditions, subjects with PD walked more slowly (F(1,22) = 16.54, p = .001, partial nu2 = .429) and with shorter steps (F(1,22) = 15.07, p = .001, partial nu2 = .406) than control subjects. In addition there were significant group by condition interaction effects for gait speed (F(2, 44) = 4.42, p = .018, partial nu2 = .167) and stride length (F(2, 44) = 4.95, p = .012, partial nu2 = .184). There was little deterioration in gait when subjects in either group carried a tray while walking compared with free walking; however, when required to carry four plastic glasses on the tray while walking, subjects in the PD group showed marked deterioration in gait speed (t(11) = 3.19, p = .009, alpha = .025) and stride length (t(11) = 3.82, p = .003, alpha = .025). Performance in the control subjects changed only marginally across the conditions., Conclusion: Subjects with moderate disability in PD experience considerable difficulty when they are required to walk while attending to a complex visuomotor task involving the upper limbs.

A new assay using a multiwell fluorescence scanner was developed for screening cytotoxicity to cells cultured in 96-well microtiter plates. The assay is based on binding of propidium iodide to nuclei of cells whose plasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measured with a multiwell fluorescence scanner increased in proportion to the number of permeabilized cells. After ATP depletion of hepatocytes and neonatal cardiac myocytes with metabolic inhibitors ("chemical hypoxia"), and exposure of Madine Darby canine kidney cells to the toxic chemical, HgCl2, propidium iodide fluorescence progressively increased. Increases of fluorescence were linearly proportional with release of lactate dehydrogenase into the culture medium. Employing this cytotoxicity screening assay, protection by various agents against lethal injury was evaluated in cultured hepatocytes during chemical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leupeptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, 1,10-phenanthroline, an inhibitor of metalloprotease, markedly protected against the onset of cell death during chemical hypoxia. Half-maximal protection after 60 min occurred at 0.5 microM. Phospholipase inhibitors, chlorpromazine (50 microM) and mepacrine (50 microM), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell killing to a lesser extent during chemical hypoxia and after oxidative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B1, did not protect against cell killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclusion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screening of potential cytoprotective agents in a variety of cell types.

We assessed the effect of acidosis on cell killing during anoxia and reoxygenation in cultured rat neonatal cardiac myocytes. After 4.5 hours of anoxia and glycolytic inhibition with 2-deoxyglucose, loss of viability was greater than 90% at pH 7.4. In contrast, at pH 6.2-7.0, viability was virtually unchanged. To model changes of pH and oxygenation during ischemia and reperfusion, myocytes were made anoxic at pH 6.2 for 4 hours, followed by reoxygenation at pH 7.4. Under these conditions, reoxygenation precipitated loss of viability to about half the cells. When pH was increased to 7.4 without reoxygenation, similar lethal injury occurred. No cell killing occurred after reoxygenation at pH 6.2. We conclude that acidosis protects against lethal anoxic injury, and that a rapid return from acidotic to physiologic pH contributes significantly to reperfusion injury to cardiac myocytes - a 'pH paradox'.

Cardiac myocytes exposed to anoxia hypercontract into a blebbed, rounded mass. Such hypercontraction is usually considered a manifestation of irreversible injury. Here, we studied functional recovery, long-term viability and ATP levels of cultured neonatal rat cardiac myocytes after metabolic inhibition with cyanide and 2-deoxyglucose, a model of 'chemical hypoxia' which mimics the ATP depletion and reductive stress of hypoxia. After addition of inhibitors, 5-day cultured myocytes ceased spontaneous contractions within 1-2 min, blebbed and hypercontracted after 35 min, and lost viability after 100 min as assessed by nuclear labelling with propidium iodide. 11-day cultured myocytes exhibited a similar progression of injury. When the metabolic inhibitors were removed, spontaneous contractions resumed after an average of 11 min in non-hypercontracted myocytes. In hypercontracted myocytes, weak contractions resumed after 35 min, regardless of the length of time spent in the hypercontracted state, but restoration of strong synchronous contractions took hours. Recovering cells remained viable and exhibited spontaneous contractions through 24 hours of observation, whereas contractility never returned in propidium iodide-labelled cells. ATP levels decreased rapidly after chemical hypoxia and were partially restored upon washout of inhibitors. ATP recovery was similar in non-hypercontracted and hypercontracted cells. Thus, contractile dysfunction during recovery in hypercontracted cells was not due to lack of regeneration of ATP. In conclusion, hypercontracture is not a manifestation of irreversible injury. Contractile dysfunction following recovery from hypercontracture observed here may represent 'in vitro stunning'.

High affinity Ins(1,4,5)P3-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins(1,4,5)P3 on intracellular Ca2+ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized sites bind Ins(1,4,5)P3 with an affinity (Kd = 7.26 +/- 0.52 nM, Hill coefficient h = 1.05 +/- 0.06) similar to that of the sites in native membranes (Kd = 6.02 +/- 1.57 nM, h = 0.99 +/- 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P3 Ins(4,5)P2, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP3 receptor will allow its further characterization, purification, and comparison of its properties with those of InsP3 receptors already purified from cerebellum and smooth muscle.

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer, with a molecular weight of 64,000 has been purified by gel filtration. The functional properties of the tetramer have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimeric enzyme. The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be different with the tetramer accumulating predominantly in the liver.

When male rats of certain strains are fed a diet with 3% or more Na saccharin, their urinary bladders develop epithelial hyperplasia and a greater incidence of tumors. Since the daily dose of saccharin is high, a link between tumor formation and the disruption of urothelial physiologic and biochemical processes has been sought. We fed male and female Sprague-Dawley rats a saccharin-free or 7.5% Na saccharin diet for 1 month. Excised bladders were mounted in flux chambers and exposed to Krebs-Ringer bicarbonate solution (KRB) or urine. Bioelectric properties and 22Na, 36Cl, and [14C]mannitol or [3H]mannitol unidirectional fluxes were measured by conventional techniques. No differences were noted between bladders from male and female animals or between Na saccharin-fed animals and animals fed the saccharin-free diet. When both surfaces of the epithelium were exposed to KRB, transepithelial dc conductance fell over 4 hr to 50% of the initial value. Conductance averaged 1.4 mS/cm2. Transepithelial potential difference (PD) was usually lumen negative and averaged 0.7 mV. Unidirectional permeability coefficients for 36Cl, 22Na, and radiomannitol were symmetric, proportional to conductance, and followed a rank order compatible with unrestricted passive diffusion. Exposure of the bladder lumen to urine from animals fed saccharin-free or Na saccharin diet hyperpolarized the transepithelial PD by more than 5 mV and raised conductance nearly threefold. Permeability coefficients remained symmetric and compatible with passive diffusion. Exposure of the lumen to solutions with the K+, Na+, and Cl concentrations and osmolality of urine simulated the conductance and PD effects of urine. We conclude that Na saccharin feeding or urine with saccharin does not uniquely affect the permeability of the excised preparation. Small hydrophilic solutes appear to cross the bladder epithelium through paracellular channels which increase in aggregate area during exposure of the lumen to urine. The hyperpolarization induced by lumenal urine is the consequence of the transepithelial K+ gradient.

An International Standard for Colistin has been established and the International Unit defined as the activity contained in 0.00004878 mg of this preparation. The unit was defined on the basis of a collaborative assay in which nine laboratories from six different countries participated. The material used to prepare the international standard had been manufactured in Japan; it was shown to have a composition similar to that of material produced in Europe and to be suitable for the assay of colistin from the existing sources of manufacture.

The study of procaine benzylpenicillin in oil with aluminium monostearate (PAM) described in this paper was undertaken to devise laboratory assay methods, based on animal tests, to evaluate those properties of PAM responsible for the persistence of penicillin in the circulation, as a preferable substitute for the existing test in man. Comparative studies of a number of batches of PAM in man proved, with statistical certainty, that the duration of the blood level of penicillin which they produced varied from batch to batch. The chemical and physical properties of the batches gave inadequate characterization and were of no value in assessing material for clinical use. A blood level duration test in rabbits was developed which reflected the blood level duration properties shown in man. The test was incorporated in the Requirements for Procaine Benzylpenicillin in Oil with Aluminium Monostearate that were set up by the WHO Expert Committee on Biological Standardization to ensure the suitability of batches for clinical use. An International Reference Preparation was necessary and was established. Batches of PAM in use in the field by WHO in anti-yaws campaigns were examined by the test described and found to be satisfactory.

The first International Reference Preparation of Viomycin was replaced by the second international reference preparation, consisting of material from the batch that provided the second Working Standard of the US Food and Drug Administration. The International Unit of viomycin was redefined as the activity contained in 0.0012285 mg of the second international reference preparation. Examination of batches of viomycin sulfate from the various sources of production showed that the second international reference preparation was suitable for their assay, whereas a sample previously proposed as the international standard of viomycin was unsatisfactory.

The second International Reference Preparation of Procaine Benzylpenicillin in Oil with Aluminium Monostearate was established in replacement of the first international reference preparation. The blood-level duration test (WHO Requirements for Biological Substances, No. 9), used to evaluate batches of manufactured material in relation to this reference preparation, was revised to relate to the new reference preparation. Thus a batch now passes the test if the lower confidence limit of the ratio is greater than 0.45, instead of 0.50-the value that related to the first international reference preparation. This change will ensure that material passing the test in the future will be of the same quality as in the past.

As supplies of the International Standard for Chlortetracycline were practically exhausted, it was replaced. The potency of the second international standard was defined on the basis of an international collaborative assay comprising 157 individual assays performed in 9 laboratories in 6 countries. The mean potencies obtained in the participating laboratories, although they varied by only 7%, were heterogeneous. However, the mean potency for all the laboratories combined did not differ significantly from that of the first international standard. The International Unit for chlortetracycline was therefore defined as the activity contained in 0.001 mg of the second international standard, corresponding to a potency of 1 000 IU/mg.

The National Institute for Medical Research, London, was requested by the WHO Expert Committee on Biological Standardization to establish a second International Standard for Oxytetracycline, since stocks of the first International Standard were depleted. A batch of 500 g of oxytetracycline dihydrate obtained in 1963 was distributed into ampoules in 125-mg amounts; the ampoules were filled with dried nitrogen and sealed. The usual procedure of exhaustively drying the material was omitted since removal of water of hydration adversely affects stability of oxytetracycline dihydrate. The proposed replacement material has been assayed biologically in terms of the first International Standard in 9 laboratories in 7 countries by means of plate-diffusion and turbidimetric assays.Significant heterogeneity of the potencies obtained by the different laboratories and within 3 laboratories was not attributable to any known differences in assay technique; the weighted mean potencies obtained by all laboratories were within a range of +/-2% of the over-all mean potency.The potency of the second International Standard is approximately 3% less than that of the first International Standard but no significant difference in composition or purity could be detected by a variety of chemical and physical analytical methods. The material has been established as the second International Standard for Oxytetracycline with a defined potency of 880 IU/mg. The International Unit of Oxytetracycline is defined as the activity of 0.0011364 mg of the second International Standard of Oxytetracycline.

The international unit of colistin methane sulfonate has been defined by collaborative assay as the activity contained in 0.00007874 mg of the international reference preparation. The definition was based on results from 7 laboratories in 5 countries which carried out assays against their existing national standards. Because of the complex heterogeneity in composition of this antibiotic the international reference preparation was not designated as an international standard.

An International Standard for Rolitetracycline has been established and the international unit of this antibiotic defined as the activity contained in 0.001004 mg of the international standard. The definition of the international unit was based on the results of a collaborative assay in which 8 laboratories in 6 different countries participated; a total of 133 assays were performed. The assay was in terms of the Working Standard of the USA Food and Drug Administration; mean potencies for individual laboratories varied within a range of only 2% of the mean for all assays although 7 different test organisms were used in both diffusion and turbidimetric assays. Individual assays, however, provided potencies that varied within a range of 40%.

The National Institute for Medical Research, London, was requested by the WHO Expert Committee on Biological Standardization to establish an International Reference Preparation of Gramicidin. This preparation was needed to standardize preparations of gramicidin containing predominantly gramicidin A, B and C, for which purpose the International Reference Preparation of Gramicidin S cannot be used. A batch of 100 g of crystalline gramicidin obtained in 1963 was distributed into ampoules in 55 mg amounts and dried in vacuo; the ampoules were then filled with dried nitrogen and sealed. The proposed international reference preparation was assayed biologically against the Master Standard of Gramicidin of the US Food and Drug Administration in 7 laboratories in 6 countries by turbidimetric methods.Significant curvature of the dose-response lines was found for most assays; no single transformation improved the linearity of assays from all laboratories. Although significant heterogeneity of potencies was obtained in 5 laboratories the mean potency ratios for all laboratories only varied over a range of 5% to 6%. The composition of the material is 7% gramicidin B, 50% gramicidin A and 25% gramicidin C; preparations of gramicidin containing appreciably higher concentrations of gramicidin B can be expected to give invalid assays against the international reference preparation.The material has been established as the International Reference Preparation of Gramicidin with a defined potency of 1000 IU/mg. The International Unit of Gramicidin is defined as the activity of 0.001 mg of the International Reference Preparation of Gramicidin.