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.      

Light and electron microscopical observations on the heterotrophic protist Thaumatomastix salina comb. nov. (syn. Chrysosphaerella salina) and its allies

The marine flagellates presently known as the chrysophytes Chrysosphaerella salina and C. tripus Takahashi & Hara have been refound and studied with light microscopy and whole mounts for electron microscopy. Based on material from Australia and Denmark (the latter from the type locality), C. salina is shown to be a colourless protist related to Reckertia sagittifera Conrad. It is characterized by a combination of flagellar features previously thought to be restricted to Reckertia , i.e. a short anterior flagellum which is scale-clad, and a longer, usually posterior but naked flagellum. The scales on the body are shown to be silicified. The new light microscopical studies have also shown considerable resemblance between C. salina, C. tripus and the genus Thaumatomastix Lauterborn. C. salina and C. tripus are therefore transferred to this genus together with Reckertia and the 2 marine species described since 1980 as belonging to Chrysosphaerella, C. triangulata Balonov and C. patelliformis Takahashi & Hara. Thaumatomastix bipartita sp.nov. is illustrated and described.
Chrysosphaerella appears to be a genus of photosynthetic, colony-forming chrysophytes restricted to freshwater habitats. Thaumatomastix is a genus of heterotrophic protists, usually solitary, which occurs in both marine and freshwater environments. The two genera show little, if any, phylogenetic relationship to each other.     

Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein.

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.     

Über einen intrazellulären Parasiten beiCryptomonas (Cryptophyceae), I

An intracellular parasite occurring inCryptomonas rostratiformis and less numerously also inC. erosa andC. phaseolus. The parasite is described. It grows in the dorsal side of the host near the nucleus from which it is optically indistinguishable in young stages. When mature the parasite fills 1/3 to 1/2 the volume of theCryptomonas cell. It is seen as a colourless blister, which pushes back the plastid of the host. Reproduction occurs by separation of the protoplast into a hundred or moreBodo-like swarmers which perhaps represent the infectious phase. Under certain conditions, however, such as during decline of theCryptomonas population, the parasite transforms into thick-walled spindle-shaped cysts. Like the swarmers these cysts are released by rupture of the cryptomonad cells. The fate of the cysts is not known. TheCryptomonas population is destroyed by the infection in the course of a few days. Literature studies have shown that the parasite has been known for a long time, but considered incorrectly by different authors as part of theCryptomonas, or as a result of phagotrophic uptake of theCryptomonas. The parasite is compared with a somewhat similar parasite inMallomonas, and with certain parasitic dinoflagellates. The similarity with the latter is superficial only as it posesses a eucaryotic nucleus. The parasite shows some similarity with the genusParadinium (Mycetozoa) as well as with certainSporozoa.

     

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.     

Phylogeny of the Bacillariaceae with emphasis on the genus Pseudo-nitzschia (Bacillariophyceae) based on partial LSU rDNA

In order to elucidate the phylogeny and evolutionary history of the Bacillariaceae we conducted a phylogenetic analysis of 42 species (sequences were determined from more than two strains of many of the Pseudo-nitzschia species) based on the first 872 base pairs of nuclear-encoded large subunit (LSU) rDNA, which include some of the most variable domains. Four araphid genera were used as the outgroup in maximum likelihood, parsimony and distance analyses. The phylogenetic inferences revealed the Bacillariaceae as monophyletic (bootstrap support ≥90%). A clade comprising Pseudo-nitzschia, Fragilariopsis and Nitzschia americana (clade A) was supported by high bootstrap values (≥94%) and agreed with the morphological features revealed by electron microscopy. Data for 29 taxa indicate a subdivision of clade A, one clade comprising Pseudo-nitzschia species, a second clade consisting of Pseudo-nitzschia species and Nitzschia americana, and a third clade comprising Fragilariopsis species. Pseudo-nitzschia as presently defined is paraphyletic and emendation of the genus is probably needed. The analyses suggested that Nitzschia is not monophyletic, as expected from the great morphological diversity within the genus. A cluster characterized by possession of detailed ornamentation on the frustule is indicated. Eighteen taxa (16 within the Bacillariaceae) were tested for production of domoic acid, a neurotoxic amino acid. Only P. australis, P. multiseries and P. seriata produced domoic acid, and these clustered together in all analyses. Since Nitzschia navis-varingica also produces domoic acid, but is distantly related to the cluster comprising the Pseudo-nitzschia domoic acid producers, it is most parsimonious to suggest that the ability of species in the Bacillariaceae to produce domoic acid has evolved at least twice.     

Long-term survival of haptophyte and prasinophyte resting stages in marine sediment

Resting stages of marine phytoplankton have been shown to have potential for long-term survival and to remain viable in marine sediments for up to about a century. This study documents for the first time long-term survival in haptophytes and prasinophytes, by germination of resting stages of Isochrysis galbana and Mantoniella squamata from up to 40-year-old sediment layers. Germination was induced by setting up sediment slurries in L1 medium at 15°C. Cyst formation was induced in culture strains acquired from the germinations by keeping mixtures of strains in the light or dark at salinities of 20 or 30. The identity of the two species was confirmed by light and electron microscopy as well as LSU and SSU rDNA-based phylogenetic analyses.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

THE MARINE DINOFLAGELLATE ALEXANDRIUM OSTENFELDII: PARALYTIC SHELLFISH TOXIN CONCENTRATION,COMPOSITION, AND TOXICITY TO A TINTINNID CILIATE1

The composition of paralytic shellfish toxins in the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen grown in unialgal culture was determined by high-performance liquid chromatography. The toxin profile revealed that the low-potency sulfamate toxin B₂ was dominant (90 molar % of total toxins), but small amounts of the weakly toxic 21-N-sulfocarbamoyl derivatives C₁₊₂ and trace amounts of the carbamate toxins GTX₂ and GTX₃ were also present. The mammalian toxicity was confirmed by a modification of the conventional AOAC mouse bioassay (0.6–1.4 pg STX_eq· cell^-1). The acute toxicity to a potential predator, the tintinnid ciliate Favella ehrenbergi (Clap, et Lach.) Jörg., was also investigated. The ciliate was able to graze on A. ostenfeldii when the cell concentration of the dinoflagellate was low (<2000 cells · mL^-1). At higher concentrations the ciliate was affected by exudates (presumably PSP toxins) that induced backward swimming followed by swelling and lysis of the cell. Fluorescence microscopy of calcofluor-stained cells was employed as an easy and rapid method to identify this and other thecate dinoflagellates.