THE ATOMIC FORCE MICROSCOPE DETECTS ATP-SENSITIVE PROTEIN CLUSTERS IN THE PLASMA MEMBRANE OF TRANSFORMED MDCK CELLS

Plasma membrane proteins are supposed to form clusters that allow ‘functional cross-talk’ between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K⁺channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be ‘glued’ with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9±0.02nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-γ-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form ‘functional clusters’ in their native environment. The ‘physiological’ arrangement of the protein molecules within a cluster requires ATP.     

The Susceptibility of Soybean Seed Lipids to Artificially-Enhanced Atmospheric Oxidation

Priestley, D. A., Werner, B. G. and Leopold, A. C. 1985. Thesusceptibility of soybean seed lipids to artificially-enhancedatmospheric oxidation.—J. exp. Bot. 36: 1653–1659. As a model system for studying possible oxidation changes insoybeans with ageing, whole soybean seeds, ground soybeans orsoybean oil were exposed to a heated oxygen atmosphere (105°C)for periods of up to 6 d. With the exception of polar lipidsof the embryonic axis, seed lipids were highly resistant tooxidative degradation provided seed structure was maintainedintact; however, the non-lipid fraction of the seed rapidlybecame discoloured. Polar lipids of ground seed material, andboth total and polar lipids in isolated oil, were less stableto oxidation than similar lipids within whole seeds. These resultsindicate that seed organization protects the lipid componentsfrom atmospheric autoxidation. Key words: Soybean, seed lipids, oxidation     

Response of Photosynthesis and Dark-CO2-Fixation to Light, CO2 and Temperature in Leaf Slices under Osmotic Stress

Photosynthesis and dark-CO₂-fixation were measured in vacuum-infiltratedleaf slices from the mesophyte Spinacia oleracea and the Mediterraneanxerophyte Arbutus unedo under hypertonic stress as a functionof light-intensity, CO₂-concentration and temperature, in theabsence of stomatal control. Under hypertonic stress, photosynthesis and dark-CO₂-fixationwere inhibited in leaf tissue from both plants. 50% inhibitionof photosynthesis in spinach occurred at about –3.0 MPa,and of dark-CO₂-fixation at about –3.5 MPa. 50% inhibitionof photosynthesis in Arbutus unedo was reached at about –4.0MPa (sorbitol as osmoticum). In both plants, osmotic dehydration decreased the slope andthe maximum of the CO₂- and light-response curves. The slopeof the CO₂-response curve of dark-CO₂-fixation was also decreasedunder hypertonic stress, but the inhibition of the maximal fixationrate was less obvious than for photosynthesis. Photosynthesis and dark-CO₂-fixation differed significantlyin their response to high temperature: under light- and CO₂-saturation,photosynthesis of spinach leaf slices had a temperature optimumat about 37 °C, and it was nearly completely inhibited at45 °C. The rate of dark-CO₂-fixation, however, increasedcontinuously up to 45 °C. Osmotic dehydration increasedthe resistance of photosynthesis to high temperatures. Key words: CO₂ response, Heat stress, Light response, Photosynthesis, Water stress     

Microtubular Structures in Macronuclei of Synchronously Dividing Tetrahymena pyriformis

SYNOPSIS. When heat-synchronized cultures of Tetrahymena pyriformis , amicronucleate strain GL, were examined by electron microscopy, intramacronuclear microtubules were observed in dividing cells. These tubules have a diameter of 180–230 A and occur either singly or packed together in bundles. They are predominantly associated with outpocketings and invaginations of the nucleus. Sections as well as negatively stained preparations of isolated macronuclear envelopes indicate that the microtubules are inserted at the inner nuclear membrane.
The findings suggest that microtubules of the spindle type participate in the process of macronuclear division.     

Mechanism of Resistance to Steroids: Glucocorticoid Receptor Defect in Lymphoma Cells

GLUCOCORTICOIDS cause cytolysis of normal and neoplastic lymphoid cells^1,2—a reaction which is one basis for the use of these compounds in the therapy of neoplastic, autoimmune and other diseases of the lymphoid system. Unfortunately, resistance to steroids often develops during treatment, rendering the hormones therapeutically ineffective.     

Caffeine Formulation for Avian Repellency

Abstract: Nonlethal management alternatives are needed to reduce avian depredation of agricultural crops. Caffeine has promise as an effective, economical, and environmentally safe avian repellent, yet formulation improvements are needed for field applications. We included sodium benzoate in subsequent formulations to enhance the solubility of caffeine. Red-winged blackbirds (Agelaius phoeniceus) discriminated between untreated rice and rice treated with 250 ppm or 10,000 ppm caffeine and sodium benzoate in captivity. We observed a positive concentration-response relationship among birds offered rice treated with 250 ppm, 1,000 ppm, 2,500 ppm, 5,000 ppm, 10,000 ppm, or 20,000 ppm caffeine and sodium benzoate. Relative to the pretreatment of the concentration-response test, repellency was >85% for rice treated with our 2,500- to 20,000-ppm treatments. We conducted seed germination experiments and associated residue analyses to evaluate the potential phytotoxicity of various caffeine formulations under controlled environmental conditions. The optimized formulation enhanced the solubility of tank mixtures and ameliorated the negative impacts of seed treatments to germination. Our caffeine formulation provides an applicable, nonlethal alternative for managing blackbird impacts to agricultural production.     

IDENTIFICATION OF LECTINS IN THE KINETIDS OF TETRAHYMENA PYRIFORMIS

Previously we described lectin-like molecules in the ciliate Tetrahymena pyriformis; by application of synthetic neoglycoconjugates it is now shown that T. pyriformis contains considerable amounts of both a β-d-glucose- and a lactose-specific lectin. No evidence for the presence of α-d-mannose-, α-d-galactose- or of α-l-fucose-specific lectins could be obtained. The two lectins, identified in T. pyriformis, are associated with the kinetids. During cell division the lectins disappear or become masked in the fission furrow. Therefore, we assume that these lectins are involved in the organization of the distribution pattern of the kinetids during cell division perhaps due to lectin—glycoprotein interactions.