High resolution spatio-temporal analysis of aquatic chemical signals using microelectrochemical electrodes

Detailed understanding of chemoreceptor cell transduction andfiltering depends on precise control and thus measurement ofthe chemical stimulus. In contrast to vision and hearing, accuratestimulus measurement in chemoreception has not been possibleat biologically relevant spatial and temporal scales. In thispaper we introduce a new high-speed (200 hz) electrochemicalmethod for the direct measurement of odor signals at biologicallyrelevant space scales (10-100 µm). We tested this systemin three applications: (i) temporal and spatial features ofodor plumes, (ii) stimulus calibrations in physiological recordingchambers and (iii) boundary layer diffusion measurements withinreceptor structures.     

Prevention of flowering and increasing sugar yield of sugarcane by application of ethephon (2-chloroethylphosphonic acid)

The inhibition of flowering in sugarcane by ethephon (2-chloroethylphosphonic acid) applied to experimental plots is well-documented; however, verification of its efficacy in large field trials is lacking. Large-scale field trials were established at Mauna Kea Agribusiness Company, Inc., a sugar and macadamia nut plantation located on the island of Hawaii, to determine whether flower inhibition attributed to ethephon would increase sugar yield. Summarization of results from 35 paired block experiments showed an 87% reduction in tasseling in the ethephon-treated blocks. The yield of sugarcane was increased by 7.5%, and the yield of sugar by 10%. The correlation (r ²) between the decrease in flowering and increase in cane and sugar yield was only 0.02 and 0.08%, respectively, indicating that the yield increase attributed to ethephon was not adequately explained by its effect on flowering. Paper No. 665 in the journal series of the Experiment Station, Hawaiian Sugar Planters' Association.     

Hydrodynamic, electron microscopic, and ligand-binding analysis of the Epstein-Barr virus/C3dg receptor (CR2)

The interaction of the Epstein-Barr virus/45-kDa proteolytic fragment of C3 (C3dg) receptor (CR2) with its viral ligand, the Epstein-Barr virus glycoprotein gp350/220, initiates the sequence of events leading to virus internalization and B lymphocyte transformation. Soluble recombinant receptor (rCR2) and gp350/220 as well as the natural ligand, C3dg, were subjected to a number of analytical techniques including gel permeation chromatography, density gradient ultracentrifugation, circular dichroism, and electron microscopy in order to determine their hydrodynamic, structural, and binding properties. Both rCR2 and gp350/220 were found to be highly extended proteins (f/fo = 2.1 and 2.4/2.2, respectively). C3dg, in contrast to the viral ligand, is only somewhat elongated (f/fo = 1.5). Soluble rCR2, visualized by high resolution electron microscopy, was shown to be an extended, highly flexible molecule comprised of ringlet domains, each approximately 24.1 A in length, which likely correspond to the short consensus repeat motif deduced from the CR2 cDNA nucleotide sequence. Ligand-binding studies carried out under physiological conditions indicated that gp350/220 binding to rCR2 was saturable and univalent, with a dissociation constant of 3.2 nM. In contrast, monomeric C3dg did not bind to rCR2 under physiological conditions; however, at reduced ionic strength, monomeric C3dg binding could be measured. These studies indicate that the affinity of the C3dg monomer for rCR2 under physiologic conditions is approximately 10(4)-fold less than that of the viral ligand. The molecular properties of rCR2 revealed in these studies provide essential information for future studies of the biologic functions of the Epstein-Barr virus/C3dg receptor.     

Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein

Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.     

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.     

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.     

Effect of recombinant murine tumor necrosis factor on hemopoietic reconstitution in sublethally irradiated mice

Intravenous bolus administration of a single 2-micrograms dose of murine rTNF-alpha to BALB/c mice 20 h before sublethal total-body irradiation (7.5 Gy) conferred significant protection against radiation-induced leukopenia. Murine rTNF-alpha administration not only reduced the decline of neutrophil and total blood cell counts after radiation, but also accelerated the subsequent normalization of peripheral blood cell counts. This was accompanied by accelerated regeneration of primitive hematopoietic progenitors, as determined by the in vivo spleen CFU assay, and the in vitro assay of the more mature hematopoietic cell compartment. This demonstrates that pretreatment with murine rTNF-alpha enhances hematopoietic reconstitution after sublethal irradiation, and indicates a possible therapeutic potential for this agent in the treatment of radiation-induced myelo-suppression.     

Expression of neural cell adhesion molecule immunoreactivity in hypertrophic myocardium

Myocardial neural cell adhesion molecule (N-CAM) is temporally regulated, being expressed during cardiac morphogenesis and innervation and suppressed in the adult heart. We have investigated the plasticity of N-CAM expression in hypertrophic muscle using the rat model of chronic hypoxia to selectively induce right ventricular hypertrophy over a 14 day time course. Sarcolemmal and intercalated disc N-CAM immunostaining was more extensive in the ventricular myocardium of hypoxic rats compared to normoxic controls. Quantitative assessment of the immunoreactivity in tissue extracts demonstrated a selective increase in the amount of N-CAM immunoreactivity in the hypertrophic myocardium of the right ventricle of rats exposed to hypoxia and this was associated with an increase of the 125 kDa isoform. We conclude that myocardial hypertrophy may be a factor influencing N-CAM expression in the heart and adhesion molecules may have a role in cardiac remodelling.     

Use of DNA purified in situ from cells embedded in agarose plugs for the molecular analysis of tk-/- mutants recovered in the L5178Y tk+/- 3.7.2C mutagen assay system

We have reported that tk-/- mutants recovered in the mouse L5178Y tk+/- 3.7.2C mutagen assay have often lost the tk+ allele. Allele loss in the tk-/- mutants is documented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk+/+ and tk+/- cell DNAs. For the routine screening of large- and small-colony tk-/- mutants DNAs for the absence of this genomic fragment, we have found that cells can be lysed in agarose plugs, and DNA of cells embedded in plugs can be purified, restricted with Nco I, electrophoresed, and analyzed on Southern blots without significant band distortion or diffusional loss of tk- specific fragments in the 2-7-kb range. Purification and restriction analysis of DNA in agarose plugs, originally developed to allow pulsed-field gel electrophoresis of very large DNA fragments, represents a convenient alternative to conventional DNA purification methods, allowing quantitative recovery of DNA from small numbers of cells, eliminating centrifugation, phenol extraction, and ethanol precipitation steps, and requiring smaller quantities of reagents.