Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO₂ uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 mol m^-2 s^-1 for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO₂ uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50–70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PA photoacoustic - PFD photon flux density - PSII photosystem II     

Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.

Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Dan?, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions among coexisting larval Odonata: an in situ experiment using small enclosures

Field experiments using small replicated enclosures focused on interactions between larval populations of Epitheca cynosura and Ladona deplanata (Odonata: Anisoptera) — two species that emerge in early spring. The presence of Epitheca reduced the total biomass of Ladona, but Ladona had no significant effect on Epitheca. These early-emerging species reduced the biomass of small instars of late-emerging Anisoptera which colonized enclosures during the experiments; and the late-emerging Anisoptera seem to have inhibited colonization by Zygoptera larvae. Results are consistent with the importance of predatory (cannibalism or mutual predation) interactions in this community.     

Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

Antibodies to tubulin were prepared in rabbits by immunization with reduced-carboxymethylated calf-brain tubulin. In immunodiffusion tests the antibodies showed full cross reactivity with the immunogen as well as with native calf-brain tubulin. The same antibodies showed cross reactivity with a factor in extract of cotton (Gossypium hirsutum L.) cotyledons but there was no full immunological identity between calf-brain tubulin and this factor. A solid-phase radioimmunoassay for quantitative estimation of this plant tubulin-like factor was developed. It measured the binding of antibodies to immobilized calf-native tubulin. Competition between the unknown soluble tubulin-like factor, and immobilized tubulin was assayed at serum dilution of 1:50. Extraction conditions which preserved the antigenic properties of the tubulin-like factor from cotton cotyledons were defined. The radioimmunoassay measured quantities of the tubulin-like factor in the range of 0.1–10 g-equivalents of calf-brain tubulin. Immediately after homogenization of the tissue only 25% of the total amount of tubulin-like activity was present in soluble form, while most of it remained in the insoluble fraction. Apparent maximal solubilization was achieved spontaneously 10 h after homogenization or by treatment with guanidine hydrochloride. These results indicate that in this material, tubulin is not released immediately by homogenization but remains assembled in microtubules and-or in a bound or sequestered form.Abbreviations NRS normal rabbit serum - RCM reduced-carboxymethylated     

Using all Gene Families Vastly Expands Data Available for Phylogenomic Inference

Traditionally, single-copy orthologs have been the gold standard in phylogenomics. Most phylogenomic studies identify putative single-copy orthologs using clustering approaches and retain families with a single sequence per species. This limits the amount of data available by excluding larger families. Recent advances have suggested several ways to include data from larger families. For instance, tree-based decomposition methods facilitate the extraction of orthologs from large families. Additionally, several methods for species tree inference are robust to the inclusion of paralogs and could use all of the data from larger families. Here, we explore the effects of using all families for phylogenetic inference by examining relationships among 26 primate species in detail and by analyzing five additional data sets. We compare single-copy families, orthologs extracted using tree-based decomposition approaches, and all families with all data. We explore several species tree inference methods, finding that identical trees are returned across nearly all subsets of the data and methods for primates. The relationships among Platyrrhini remain contentious; however, the species tree inference method matters more than the subset of data used. Using data from larger gene families drastically increases the number of genes available and leads to consistent estimates of branch lengths, nodal certainty and concordance, and inferences of introgression in primates. For the other data sets, topological inferences are consistent whether single-copy families or orthologs extracted using decomposition approaches are analyzed. Using larger gene families is a promising approach to include more data in phylogenomics without sacrificing accuracy, at least when high-quality genomes are available.     

Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi‐quantitative approaches to their interpretation.     

A Durable Alternative for Proton‐Exchange Membranes: Sulfonated Poly(Benzoxazole Thioether Sulfone)s

To develop a durable proton‐exchange membrane (PEM) for fuel‐cell applications, a series of sulfonated poly(benzoxazole thioether sulfone)s ( SPTESBOs) are designed and synthesized, with anticipated good dimensional stability (via acid–base cross linking), improved oxidative stability against free radicals (via incorporation of thioether groups), and enhanced inherent stability (via elimination of unstable end groups) of the backbone. The structures and the degree of sulfonation of the copolymers are characterized using Fourier‐transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy (¹H NMR and ¹⁹F NMR). The electrochemical stabilities of the monomers are examined using cyclic voltammetry in a typical three‐electrode cell configuration. The physicochemical properties of the membranes vital to fuel‐cell performance are also carefully evaluated under conditions relevant to fuel‐cell operation, including chemical and thermal stability, proton conductivity, solubility in different solvents, water uptake, and swelling ratio. The new membranes exhibit low dimensional change at 25°C to 90°C and excellent thermal stability up to 250°C. Upon elimination of unstable end groups, the co‐polymers display enhanced chemical resistance and oxidative stability in Fenton's test. Further, the SPTESBO‐HFB‐60 (HFB‐60=hexafluorobenzene, 60 mol% sulfone) membrane displays comparable fuel‐cell performance to that of an NRE 212 membrane at 80°C under fully humidified condition, suggesting that the new membranes have the potential to be more durable but less expensive for fuel‐cell applications.     

A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria.

The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.