Secretion and export of IGF-1 in Escherichia coli strain JM101

Summary The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.     

Predator driven changes in community structure

Summary The zooplankton community of a small pond changed markedly with temporal variation in predation pressure. Long term changes in zooplankton community structure occurred following the replacement of planktivorous fish by phantom midge (Chaoborus americanus) larvae as the predominant predator of zooplankton. The interannual changes following the establishment of Chaoborus included the apparent or near extinction, of species ill adapted to the new predation pressure and the successful colonization of well adapted species. Seasonal changes in the species composition and size distribution of the zooplankton community correlate with temporal variation in predation intensity associated with temperature-activity patterns of the predator or changes in the stage structure of the predator population.     

Associational plant refuges: convergent patterns in marine and terrestrial communities result from differing mechanisms

Summary An associational plant refuge occurs when a plant that is susceptible to herbivory gains protection from herbivory when it is associated with another plant. In coastal North Carolina, the abundance of the palatable red alga Gracilaria tikvahiae is positively correlated with the abundance of the unpalatable brown alga Sargassum filipendula during times of increased herbivore activity. To see if grazing by the sea urchin Arbacia punctulata could generate this pattern, controlled experiments were conducted in out-door microcosms and in the laboratory. Gracilaria beneath a canopy of Sargassum was eaten significantly less than Gracilaria alone. When Arbacia were excluded, Gracilaria alone grew significantly more than Gracilaria beneath Sargassum, demonstrating that Sargassum is a competitor of Gracilaria. Experiments investigating Sargassum's deterrent role indicated that Sargassum decreased the foraging range of Arbacia and the rate at which it fed on Gracilaria. Additional experiments with plastic Sargassum mimics indicated that the decreased grazing on Gracilaria was not a result of Sargassum morphology, but was probably attributable to some chemical characteristic of Sargassum. The pattern of increased grazing in monocultures (only Gracilaria present) versus polycultures (both Gracilaria and Sargassum present) demonstrated in this study also has been demonstrated for plant-insect interactions in terrestrial communities. In these communities, insect density is higher in monocultures than in polycultures because insects find and immigrate to monocultures more rapidly, and once in a monoculture, they emigrate from them less often than from polycultures. In this study, urchins did not find and immigrate to monocultures more rapidly, nor did they tend to stay in them once they were found; in fact, they emigrated from monocultures of Gracilaria more rapidly than from Gracilaria and Sargassum polycultures. Increased grazing in Gracilaria monocultures resulted from increased rates of movement and feeding of individual herbivores, not from increased herbivore density as has been reported for terrestrial systems.     

Respiration and soluble sugar metabolism in sugar pine embryos

Embroys excised from dormant seeds of sugar pine ( Pinus lambertiana Dougl.) incubated at 25°C (non-dormancy-breaking) or stratified at 5°C (dormancy-breaking) were analyzed to determine temperature effects on the relative activities of respiration and fermentative metabolism, the levels of soluble sugers and the activities of the hydrolytic enzymes, invertase and sucrose synthase, as related to the release of dormancy and germinatio. At 25°C, despite a sharp drop in embryo oxygen uptake after 48 h, a simultaneous decline in acetaldehyde and ethanol concentrations indicated that there was not a shift to fermentative metabolism. The concentrations of soluble sugars showed no treatment effects. Embryo invertase (EC 3.2.1.26) activity changed only slightly at either temperature, while stratification was accompanied by a 4-fold increase in sucrose synthase (EC 2.4.1.13) activity (cleavage direction). Upon transfer of stratified seeds to 25°C, embryo sucrose synthase activity rapidly increased almost 10-fold, with the increase beginning prior to germination, while mvertase activity increased 20-fold, concomitant with germination.     

Role of Magnesium in the Binding of Substrate and Effectors to Phosphoenolpyruvate Carboxylase from a CAM Plant

The binding of phosphoenolpyruvate, malate, and glucose 6-phosphate to phosphoenolpyruvate carboxylase purified from Crassula argentea Thunb. was measured using both the intrinsic tryptophan fluorescence of the enzyme and the extrinsic fluorescence of the complex of 8-anilino-1-napthalenesulfonate with the enzyme. It was found that the substrate phosphoenolpyruvate can bind in the absence of magnesium but is bound in greater quantities and more tightly when magnesium is present. Malate reduces the binding of phosphoenolpyruvate, while glucose 6-phosphate increases the binding of the substrate. Glucose 6-phosphate requires magnesium to bind to the enzyme, while malate does not. The general trends from the binding experiments using fluorescence methods were confirmed by activity determinations using assays performed in the absence of magnesium.     

Effect of a Longitudinally Applied Voltage Upon the Growth of Zea mays Seedlings

The electrical parameters that affect young seedling growth were investigated. Voltages ranging from 5 to 40 volts were applied longitudinally along the mesocotyl region of 4-day old Zea mays L. (cv Silver Queen) seedlings for periods of 3 or 4 hours. It was determined that: (a) making the tips of the seedlings electrically positive relative to the base strongly inhibited shoot growth at 5 volts, whereas the reverse polarity had no effect; (b) at higher voltages, making the tip of the seedlings negative caused less growth inhibition than the reverse polarity at each voltage level; (c) the higher the applied voltage the greater the degree of inhibition; and, (d) the more growth inhibition experienced by the plants the poorer, and slower, their recovery. Previous observations of a relationship between the amount of free indole-3-acetic acid in the mesocotyl cortex and the growth rate of the mesocotyl and of gravitropism-induced movement of labeled indole-3-acetic acid from the seed to the shoot lead to the prediction of a voltage-dependent gating of the movement of indole-3-acetic acid from the stele to the cortex. This provided the basis for attempting to alter the growth rate of seedlings by means of an applied voltage.     

Heterogeneity in lymphocyte spectrin distribution: ultrastructural identification of a new spectrin-rich cytoplasmic structure

Spectrin-like proteins are found in a wide variety of non-erythroid cells where they generally occur in the cell cortex near the plasma membrane. To determine the intracellular distribution of alpha-spectrin (alpha-fodrin) in lymphocytes, we have developed an immunoperoxidase method to localize this protein at the ultrastructural level. Of considerable interest, particularly with regard to our efforts to determine the function of spectrin in this cell type, was the finding that its subcellular localization and its relationship with the plasma membrane can vary dramatically. Based on its position in the cell, alpha-spectrin can occur in two forms in lymphocytes: one that associates closely with the plasma membrane and another that occurs at some distance from the cell periphery, either as a single large aggregate or as several smaller ones. The single large aggregate of spectrin is a stable feature in a number of lymphocyte cell lines and hybrids which were used to examine its ultrastructural characteristics. A previously undescribed cellular structure, consisting of a meshwork of spectrin filaments and membranous vesicles, was identified in these cells. This structure could be induced to dissipate in response to membrane perturbants (e.g., hyperthermia and phorbol esters, known effectors of lymphocyte function and differentiation) and the patterns resulting from the redistribution of spectrin were a reflection of those observed routinely in lymphocytes in situ. The correlation between naturally occurring spectrin localization patterns and those seen after membrane perturbation suggested the possibility that spectrin distribution is indicative of particular maturation stages or functional states in lymphocytes. The implications of these findings with regard to the role of spectrin in lymphocyte function are discussed.     

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased