Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Molecular genetics in clinical practice: evolution of a DNA diagnostic service.

The development of a molecular genetics diagnostic service over a three year period was studied in a National Health Service region with a population of three million. Starting from a time when few diagnostic applications were possible, the number of disorders and the overall demand had grown rapidly. Conditions for which molecular genetic diagnosis had been provided included Duchenne and Becker muscular dystrophy, myotonic dystrophy, Huntington''s disease, and cystic fibrosis. Of 405 requests for diagnosis, 151 (37%) related to determination of carrier state, 187 (46%) to determining the feasibility of future prenatal diagnosis, and 67 (17%) were prenatal diagnostic biopsy samples, almost exclusively of first trimester chorion. DNA samples for future diagnostic use with a wide range of genetic disorders had also been banked. The study showed a need for close integration with clinical genetics services to allow satisfactory genetic counselling and interpretation of risks.     

Induction and release of secondary seed dormancy in genetically pure lines of Avena fatua

Induction and release of secondary dormancy in genetically pure dormant (AN-51, Mont 73) and non-dormant (CS-40, SH-430) lines of wild oat ( Avena fatua L.) were studied. These lines differed with regard to the optimal period of anaerobiosis necessary for induction of dormancy, and/or the degree (% of seeds acquiring dormancy) and duration of the dormancy induced. Secondary dormancy could be induced more effectively in the after-ripened seeds of dormant lines than in the non-dormant lines, where only a short-term dormancy could be induced (in 5–7 week-old-seeds). Higher anaerobiosis temperatures were more effective in inducing dormancy in all lines studied. Thus, as with primary dormancy, wild oat biotypes exhibit genetic variability in their secondary dormancy behaviour and factors like temperature can modify the expression of this trait.
The germination stimulants kinetin, isopentenyl adenine, sodium azide, potassium nitrate, ethanol and substituted phthalimides, which break primary dormancy in wild oats, stimulated germination of secondarily dormant seeds (line AN-51). Since these chemicals are structurally diverse, primary and secondary dormancies appear to be similar in part in their regulation.
Salicylhydroxamic acid, an inhibitor of cyanide-insensitive (alternative) respiration, did not inhibit: 1, spontaneous release of secondary dormancy in the line SH-430; and 2, stimulation of germination of secondarily dormant AN-51 seeds by various chemicals (except azide), suggesting that this respiratory pathway is not necessary for the release of induced dormancy.     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

Uniconazole-induced thermotolerance in soybean seedling root tissue

Soybean [Glycine max(L.) Merr. cv. A2] seeds were germinated in 0 or 1 mg 1¹ (3.4 uM) uniconazole, after which seedling roots were excised and exposed to 22 or 48°C for 90 min. Prior to the temperature treatments there were few ultrastructural differences between uniconazole-treated seedling roots and the controls. Following exposure to 48°C, electron micrographs revealed near complete loss of normal ultrastructure in control epidermal root cells, whereas cellular integrity was maintained in treated roots, indicating that uniconazole conferred tolerance to high temperature. Total electrolyte, sugar and K⁺ leakage were all greater from control roots than treated roots during exposure to 48°C. Proline content in the roots was unaffected by uniconazole at 22°C but was 25–30% greater in treated tissue than in controls following exposure to 48°C. Malondialdehyde content was unaffected by uniconazole at 22°C but was nearly 20% less in treated tissue than in controls following high temperature exposure. This indicates that uniconazole decreased high-temperature-induced lipid peroxidation. Uniconazole elevated several antiox-idant systems in the roots, including water-soluble sulfhydryl concentration and catalase, peroxidase and superoxide dismutase activities. These findings are consistent with the hypothesis that uniconazole-induced stress tolerance is due, at least in part, to enhanced antioxidant activity which reduces stress-related oxidative damage to cell membranes.     

On the Estimation of the Population Mean with Known Coefficient of Variation

For the estimation of population mean a class of estimators has been proposed when the coefficient of variation is known and its efficiency is compared with the usual unbiased estimator and the estimators suggested by various researchers. The properties of the proposed class of estimators have been also discussed in the case of normal population.     

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm² tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.