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.      

Differences in mortality from acute myocardial infarction between coronary care unit and medical ward: treatment or bias?

To analyse the effectiveness of coronary care units in reducing mortality from myocardial infarction 18 hospitals ranging from large urban teaching hospitals to small country hospitals were stratified into four levels of care. Previous analysis had failed to show significant differences in the overall mortality in hospital among levels. There were significant differences in mortality, however, between those patients allocated to be cared for in the coronary care unit and those in the medical wards in the more advanced hospitals. The differences were largest in the hospitals with the most elaborate facilities (level 1) and non-existent in those with the least (level 4). Several analytical approaches to these observed differences indicated that they were: (a) reduced by adjustment for age and severity of infarction; (b) paralleled by differences in coexisting disease recorded on death certificates; (c) no longer significant at level 1 after allowing for differences in coexisting disease; and (d) not significant at any level after exclusion of patients first diagnosed at necropsy. These findings suggest that the observed differences in mortality between coronary care units and medical wards are largely due to bias in selection and diagnosis.     

Evidence for the uninemy of eukaryotic chromatids

Polytene chromosomes of Chironomus thummi were stretched to their rupture in a pronase solution. A 176±26-fold elongation was achieved. The DNA compaction ratio, defined as the ratio of DNA length in a haploid set (85±5 mm) to the length of a polytene chromosome set (520±40 m), was 164±22. Closeness of these two values demonstrates the uninemy of the chromatids of Chironomus chromosomes. The effect of ethidium bromide on the elastic properties of chromosomes prestretched in a pronase solution and the lengthening of these chromosomes after ethidium staining suggest that DNA molecules are double-stranded and supercoiled to the moment of the chromosome rupture. It is concluded that a Chironomus chromatid consists of a single DNA molecule (or of a single chain of linked DNA molecules) both ends of which are located in the telomeres.To the memory of Prof. Vera V. Khvostova     

Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations

Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase.     

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.     

Photoperiodic reaction in the beet webworm Loxostege sticticalis L. (Pyraloidea, Crambidae) from eastern and western parts of its Eurasian range

The beet webworm, Loxostege sticticalis L., is a very dangerous polyphagous insect pest whose outbreaks periodically occur in southern Russia and northern China. The aim of our work was to describe the photoperiodic response of beet webworm populations from western (Krasnodar Territory and Rostov Province) and eastern [Buryatia and China (Hebei Province)] parts of the pest range. The insects were reared under constant photoperiods of 12–18 h and constant temperatures of 19–25°C. Incidence of diapause at different photoperiods did not show any considerable geographic differences, and the critical day length at which 50% of prepupae arrested their development was about 14–15 h in all the populations studied at experimental temperatures. The results obtained agree with the hypothesis on the existence of an area (or areas) where the pest survives during adverse periods and wherefrom it spreads when an outbreak begins (Saulich et al., 1983; Goryshin et al., 1986). Presumably, the larger part of the pest native habitats (to the north of the steppe zone) is occupied by temporary populations, incapable of surviving for long periods without an inflow of migrants from more southerly steppe regions.     

Photo-thermal regulation of diapause in Trichogramma piceum Djur. (Hymenoptera, Trichogrammatidae)

Interaction of the photoperiodic conditions of development of maternal females (day lengths of 2 to 22 h at 20°C) with the thermal regime of development of their progeny (temperature of 12 to 15°C at day length of 12 h) in determination of prepupal diapause in Trichogramma piceum was studied under laboratory conditions. At 15°C the diapause was practically absent. At lower temperatures, the proportion of diapausing prepupae was maximal (25% of larvae at 14°C, 70% of larvae at 13°, and 80% of larvae at 12°C) if the maternal females developed under short day conditions (10–12 h). When maternal females developed at day lengths of 18–20 h, diapause was rarely recorded at all temperatures, while ultra-short (less than 8–10 h) days also caused a decrease in the proportion of diapausing progeny. The right (ecologically important) threshold of this maternal long-day photoperiodic response was about 14–15 h independently of the temperature during the progeny development. These results make it possible to clarify the mechanism of the “maternal photoperiodic correction of the progeny thermal response.” Although the impact of the maternal photoperiodic response can be revealed only within a very narrow thermal range, the relative strength of the diapause-inducing effect of different day lengths is independent of the temperature regimen of the progeny development.     

Photoperiodic responses of pupa and adult determining maternal influence on progeny diapause in two species of the genus Trichogramma (Hymenoptera, Trichogrammatidae)

The adult photoperiodic responses of two Trichogramma species were investigated by exposing adults during 3 days to one of the seven light: dark regimes: L: D = 4: 20, 8: 16, 12: 12, 14: 10, 16: 8, 18: 6, and 20: 4. The preimaginal stages of these individuals developed under short (L: D = 12: 12), long (L: D = 20: 4) or intermediate photoperiods (L: D = 14: 10 and L: D = 16: 8 for Trichogramma principium and T. embryophagum, respectively). The progeny of these females developed under short day L: D = 12: 12 and at two moderately diapause-inducing temperatures (13 and 14°C for T. principium, 14 and 15°C for T. embryophagum). In both Trichogramma species which developed at both temperatures, the percentage of diapausing prepupae was significantly dependent both on the photoperiodic conditions of the preimaginal development of the maternal generation and on the photoperiod which influenced the adult females. The adults showed a typical long-day photoperiodic response with a threshold day length of ca 13 h in T. principium and ca 16 h in T. embryophagum, which practically coincided with the thresholds of the pupal photoperiodic responses of these species revealed in our previous studies. However, the ultra-short photoperiods (L: D = 4: 20 and 8:16) caused a relatively stronger diapause-inducing effect on the progeny when applied to the adult females than when it was applied to the pupae. Thus, in both the Trichogramma species studied, the patterns of photoperiodic responses of pupa and adult were somewhat different although they almost coincided in the “ecologically significant” part of the photoperiodic scale.     

Effects of cold shock on host egg parasitization by females of Trichogramma buesi Voegele (Hymenoptera, Trichogrammatidae)

The effects of cold shock (4°C for 18–24 h) on survival of Trichogramma buesi females, their fecundity, and the inclination to parasitize grain moth eggs were studied under laboratory conditions. Cold shock did not result in any significant change in the survival rate, whereas the fraction of females that parasitized grain moth eggs and the fecundity of these females slightly decreased. However, females which had already started oviposition before cold shock infested grain moth eggs much more frequently. In most insect species studied, cold shock results in a sharp decrease or even disruption of the effect of experience on the subsequent behavior. Thus, the results of this study suggest that “the effect of acquired experience” (a tendency to continue infestation of the particular host species) in Trichogramma females is based not on learning or not only on learning but on some other, possibly hormonal mechanisms.